CN113846173A

CN113846173A - Novel target, primer group and detection method for cronobacter sakazakii detection

Info

Publication number: CN113846173A
Application number: CN202111021185.0A
Authority: CN
Inventors: 满朝新; 姜毓君; 付世骞; 杨鑫焱; 秦雪; 宋丹靓敏
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2021-09-01
Filing date: 2021-09-01
Publication date: 2021-12-28

Abstract

A new target, a primer group and a detection method for detecting cronobacter sakazakii belong to the technical field of food safety. In order to solve the problems of complicated steps, long time consumption, low detection efficiency, small quantity of related genes for nucleic acid detection and weak specificity of the traditional detection method of the cronobacter sakazakii, the invention provides a novel target for detecting the cronobacter sakazakii, an LAMP primer is designed by utilizing the target, the sample DNA is taken as a substrate for LAMP amplification reaction, HNB dye is added into a system before the reaction, after the amplification is carried out for a period of time under a constant temperature condition, whether target bacteria exist in the sample can be judged by observing color change through naked eyes, the visual rapid detection of the amplified cronobacter sakazakii is realized, and the detection method has higher sensitivity, specificity and practicability and has wide application prospect.

Description

Novel target, primer group and detection method for cronobacter sakazakii detection

Technical Field

The invention belongs to the technical field of food safety, and particularly relates to a new target, a primer group and a detection method for cronobacter sakazakii detection.

Background

The cronobacter sakazakii is one of cronobacters, is easy to cause a series of diseases of newborns, has high lethality and is a very important food-borne pathogenic bacterium. The cronobacter sakazakii can pollute various dairy products, meat products, eggs and fruits and vegetables, wherein infant formula milk Powder (PIF) is one of the main pollution routes, which makes the bacteria the subject of important precaution and monitoring in the milk powder processing industry. The currently available methods for detecting cronobacter sakazakii in food mainly include conventional microorganism culture methods, immunological detection methods and molecular biological detection methods. The traditional detection method is based on a plate counting method, the detection period is generally 5-7 days, the labor capacity is large, the specificity is not high, and false positive detection is easy to cause. The immunological detection method usually requires expensive equipment and long detection time, and cannot meet the requirement of rapid detection. Compared with the traditional culture method, the detection method developed based on the nucleic acid technology becomes a hotspot for rapidly detecting the bacillus, and the search for the specific nucleic acid target of the cronobacter sakazakii is the core of the development of the related detection technology.

Much research is currently directed to specific nucleic acid targets of the genus Cronobacter, including: 16S rRNA gene, 23S rDNA gene, transfer tRNA-Glu gene, dnaG gene inside a polymer synthetic operon, alpha-glucosidase (gluA) gene, outer membrane protein A (ompA) gene, zinc-containing metalloprotease (zpx) gene, 16S-23S rDNA Internal Transcription Spacer (ITS) gene sequence, wehC gene, wzx gene, and the like. However, the related genes for nucleic acid detection of cronobacter sakazakii are small in number and low in specificity, so that the cronobacter sakazakii cannot be effectively distinguished from other strains of cronobacter and some enterobacteriaceae with close relativity, and the detection sensitivity needs to be improved. Therefore, a new nucleic acid target with high specificity, high stability and wide applicability is screened, and a method for detecting the cronobacter sakazakii with high speed, high efficiency and strong specificity is established, so that the method has very important significance for ensuring the safety of the infant formula food.

Research shows that sialic acid from breast milk or PIF can be used as a nutrient source and colonized in human intestinal tracts, during the evolution process, the cronobacter sakazakii is the only cronobacter sakazakii with the coding of nanAKT gene cluster utilizing sialic acid, and the metabolic function can provide convenience for infecting newborns.

In recent years, the application range of the loop-mediated isothermal amplification (LAMP) technology is gradually widened due to the characteristics of simplicity, rapidness, low detection limit and the like, so that the LAMP technology has a great development prospect, and the LAMP technology combined with a visual detection method is gradually one of effective means for rapidly detecting food-borne pathogenic bacteria. The hydroxynaphthol blue (HNB) dye method is a method in which the color changes according to the change of the pH value of the solution, so that the amplification result can be observed and judged by naked eyes. The whole process avoids uncovering detection, so that false positive results can not be generated, special detection equipment is not needed, the method is economical and practical, is suitable for on-site rapid detection, and is widely concerned and applied in recent years by combining with LAMP technology.

Disclosure of Invention

The invention provides application of a section of nucleotide sequence in detecting the cronobacter sakazakii, aiming at solving the problems of complicated steps, long time consumption, low detection efficiency, small quantity of related genes for nucleic acid detection and weak specificity of the traditional detection method of the cronobacter sakazakii, wherein the nucleotide sequence is shown as SEQ ID No. 1.

The invention also provides a primer group for detecting cronobacter sakazakii, which is designed according to the nucleotide sequence and specifically comprises the following primers:

outer primer F3: 5'-ATCTCGCTGCCCGCTATC-3', respectively;

outer primer B3: 5'-GTATGCTCCCAGTACGTGTC-3', respectively;

inner primer FIP: 5'-ACTGATGCGATTTACGACGGGTCACGGTGAATGATGCCCA-3', respectively;

the inner primer BIP: 5'-GGCGCTTCGGTATGTCCTGGTCTCCCGGTTCCCTGATC-3' are provided.

The invention also provides a method for visually detecting cronobacter sakazakii, which is characterized by comprising the following steps:

s1, extracting the DNA of the sample to be detected by using the DNA extracting solution;

s2, establishing an LAMP system of Cronobacter sakazakii by taking the sample DNA obtained in S1 as a template, and adding HNB dye for amplification;

s3, observing color change, if the amplification product is changed from violet to sky blue, indicating that the sample to be detected contains cronobacter sakazakii, and if the amplification product is in violet and does not change, indicating that the sample to be detected does not contain cronobacter sakazakii.

Further limiting, the composition of the DNA extracting solution of S1 is Chelex-1005g in bead shape, 401mL in NP and 19mL in TE solution, and the pH value is 8.0-9.0; the TE solution was prepared from 10mM Tris-HCl and 1mM EDTA.

Further limited, the DNA extraction method described in S1 is as follows: taking 300 mu L-1 mL of sample to be detected in a 2mL EP tube, centrifuging at 4 ℃ and 12000rpm for 2min, discarding supernatant, adding 300 mu LDNA extracting solution into the precipitate, uniformly mixing, incubating at 56 ℃ for 30min, carrying out water bath at 90-100 ℃ for 8 min-10 min, centrifuging at 4 ℃ and 12000rpm for 2min, and taking supernatant as DNA.

Further, the LAMP system of Cronobacter sakazakii of S2 has a total volume of 25 μ L, and comprises the following components: the primer set, dNTP and Mg²⁺10 × ThermoPol Buffer, Bst 2.0WarmStart DNA polymerase and template DNA; the concentration ratio of the outer primer to the inner primer in the primer group is 1:8, the final concentration of dNTP is 1.8 mM-2.2 mM, and Mg²⁺The final concentration is 3-5 mM, the addition amount of 10 × ThermoPol Buffer is 2.5 μ L, the initial concentration of Bst 2.0WarmStart DNA polymerase is 8000U/mL, the addition amount is 0.8 μ L, and the addition amount of template DNA is 2 μ L.

Further defined, the final concentration of the HNB dye in S2 is 120-180 mu M.

Further, the temperature of the amplification reaction in S2 is 62 ℃ to 64 ℃.

Further, the time of the amplification reaction in S2 is 30-60 min.

Further, when the sample is milk powder in S1, the sample pretreatment method is as follows: dissolving 10g of milk powder in 90mL of sterile peptone water solution, mixing uniformly, centrifuging at 4 ℃ and 5000rpm for 5min, and discarding the supernatant; the pellet was re-solubilized in peptone water solution for a second centrifugation, added with BPW re-solubilization and used for DNA extraction.

The principle of the detection method of the invention is as follows: the LAMP technology utilizes 4 primers to generate a product with a special stem-loop structure at about 65 ℃ through automatic cycle strand displacement reaction under the action of Bst DNA polymerase, and can generate a target gene sequence with 109 copy number within 1h, and finally, DNA fragments with different molecular sizes are generated to be LAMP products, so that the aim of rapidly amplifying target nucleic acid fragments is fulfilled. HNB is a metal ion indicator, combines with magnesium ions to make the initial color of a reaction system be violet, and as the reaction proceeds, Mg²⁺Reacting with the separated pyrophosphate ions to generate magnesium pyrophosphate precipitate, wherein HNB loses the magnesium ions to change the color of the system into sky blue, and the unreacted system still maintains the violet color.

The invention has the beneficial effects that:

1) according to the invention, the specific nucleic acid target nanC of the cronobacter sakazakii is used as a target fragment for the first time to design the LAMP primer with high matching, so that other strains with close relativity to the cronobacter sakazakii can be effectively distinguished on a molecular level, and a theoretical basis is provided for a molecular detection method for distinguishing each strain in the cronobacter sakazakii based on a nucleic acid technology. Meanwhile, a foundation is laid for the specificity and sensitivity detection of the strain in PIF and other foods.

2) The LAMP technology and the visualization method are combined, the operation is simple and convenient, the result can be observed by naked eyes through the color change of the HNB dye, and meanwhile, the amplified product is judged without uncovering for detection, so that the influence of aerosol products in the air on the result is avoided, and the convenience and the accuracy of the detection are effectively improved.

3) The invention greatly reduces the total detection time on the premise of ensuring the sensitivity. The DNA extraction process is about 50min, only LAMP amplification steps are needed in the detection process, the result can be observed within 30min at the shortest time, the detection of a plurality of samples can be simultaneously carried out, the foundation is laid for the development of a rapid detection kit of the Cronobacter sakazakii and a multiple detection technology based on a nucleic acid technology, and meanwhile, the visualization method established by the invention can be applied to the detection of more food samples or other pathogenic bacteria.

Drawings

FIG. 1 is a graph showing the effect of the concentration ratio of the outer primer to the inner primer on the LAMP reaction; in the figure, M is DNAmarker, 1 is negative control, and 2-7 are ultraviolet light electrophoresis bands corresponding to the final concentration ratios of the outer primer to the inner primer of 1:2, 1:4, 1:6, 1:8, 1:10 and 1:12 respectively;

FIG. 2 is a graph showing the effect of dNTP concentration on LAMP reaction; in the figure, M is DNAmarker, 1 is negative control, and 2-8 are corresponding ultraviolet light electrophoresis bands when the dNTP final concentration is 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 and 2.4mM respectively;

FIG. 3 is a graph showing the effect of magnesium ion concentration on LAMP reaction; in the figure, M is DNAmarker, 1 is negative control, and 2-7 are respectively Mg²⁺Electrophoresis bands under ultraviolet light corresponding to final concentrations of 1, 2, 3, 4, 5 and 6 mM;

FIG. 4 is a graph showing the effect of Bst 2.0DNA polymerase addition amount on LAMP reaction; in the figure, M is a DNA Marker, 1 is a negative control, and 2-7 are corresponding ultraviolet light electrophoresis bands when the addition amount of the enzyme is 0.6, 0.7, 0.8, 0.9, 1.0 and 1.1 mu L respectively;

FIG. 5 is a graph showing the effect of reaction temperature on LAMP reaction; in the figure, M is DNAmarker, 1 is negative control, and 2-8 are corresponding ultraviolet light electrophoresis strips at the reaction temperatures of 60, 61, 62, 63, 64, 65 and 66 ℃ respectively;

FIG. 6 is a graph showing the effect of amplification time on LAMP reaction; in the figure, M is DNAmarker, 1 is negative control, and 2-6 are corresponding ultraviolet light electrophoresis bands amplified for 30min, 45 min, 60min, 75 min and 90min at the temperature of 65 ℃;

FIG. 7 is a graph of the optimization result of the addition amount of HNB in the LAMP system; in the figure, M is DNAmarker, 1 is negative control, and 2-5 are corresponding ultraviolet light electrophoresis bands when the HNB dye mother liquor is added to make the final concentration respectively 90, 120, 150 and 180 mu M;

FIG. 8 is a graph showing the result of detection sensitivity of the LAMP-HNB method for a pure culture of Cronobacter sakazakii; in the figure, M is DNAmarker, 1 is negative control, and 2-9 are concentration of 4.5 × 10 for pure culture of cronobacter sakazakii respectively⁶CFU/mL、4.5×10⁵CFU/mL、4.5×10⁴CFU/mL、4.5×10³CFU/mL、4.5×10²CFU/mL, 4.5X 10CFU/mL, and 4.5X 10^-1Electrophoresis strip under the corresponding ultraviolet light in CFU/mL;

FIG. 9 is a graph showing the result of the LAMP-HNB method on the detection sensitivity of Cronobacter sakazakii in infant formula; in the figure, M is DNAmarker, 1 is negative control, and 2-9 are respectively the concentration of cronobacter sakazakii polluted in the infant formula milk powder of 5.7 multiplied by 10⁶CFU/mL、5.7×10⁵CFU/mL、5.7×10⁴CFU/mL、5.7×10³CFU/mL、5.7×10²CFU/mL, 5.7X 10CFU/mL, and 5.7X 10^-1CFU/mL。

Detailed Description

The present invention will be described in further detail with reference to the following examples and drawings, and reagents and equipment used in the present invention are commercially available.

The names and sources of the strains used in the following examples are shown in table 1.

Table 1 examples used the strain name and source

Example 1: screening of new target of cronobacter sakazakii and design of primer group

Whole genome sequence alignment of Cronobacter sakazakii was performed by the BLAST method (https:// BLAST)NCBI. nlm. nih. gov/BLAST. cgi) looking for their consensus genomic sequence, while these nucleic acid fragments were not present in the other 6 cronobacters and non-cronobacters in the BLAST alignment at NCBI. The cronobacter sakazakii is the only species in the cronobacter sakazakii which can grow by using exogenous sialic acid as a carbon source, because the cronobacter sakazakii is the only cronobacter sakazakii which is encoded by nanAKT gene cluster using sialic acid, and in the screened specific nucleic acid fragment, the nanC gene belongs to the nanAKT gene cluster, therefore, the nanC gene is selected as a new target of the cronobacter sakazakiiThe nucleus of the nanC gene The nucleotide sequence is shown as SEQ ID No. 1.

LAMP primer design is carried out on the screened specific nucleic acid gene nanC through online software PrimeExplorer V5(http:// PrimeExplorer. jp/lampv5e/index. html), BLAST comparison is carried out on the specific nucleic acid gene nanC, the optimal specific primer is screened out for LAMP amplification, and the set with the best amplification efficiency is used for subsequent experiments.

The obtained primer set specifically contained the following primers:

outer primer F3: the nucleotide sequence is shown as SEQ ID No.2 (5'-ATCTCGCTGCCCGCTATC-3');

outer primer B3: the nucleotide sequence is shown as SEQ ID No.3 (5'-GTATGCTCCCAGTACGTGTC-3');

inner primer FIP: the nucleotide sequence is shown as SEQ ID No.4 shows (5' -ACTGATGCGATTTACGACGGGTCACGG) TGAATGATGCCCA-3’)；

The inner primer BIP: the nucleotide sequence is shown as SEQ ID No.5 shows (5' -GGCGCTTCGGTATGTCCTGGTCTCCCG) GTTCCCTGATC-3’)。

Example 2: method for visually detecting cronobacter sakazakii

S1, extracting DNA of sample to be detected by using DNA extracting solution

1) Preparing a DNA extracting solution: 5g of bead-shaped Chelex-100 was weighed in an Erlenmeyer flask, and 1mL of NP 40 and 19mL of TE solution were added thereto to prepare 20mL of a DNA extract solution having a pH of 8.0 to 9.0. The preparation method of TE comprises the following steps: 10mM Tris-HCl, 1mM EDTA.

2) DNA extraction: taking 300 mu L-1 mL of sample to be detected in a 2mL EP tube, centrifuging at 4 ℃ and 12000rpm for 2min, discarding supernatant, adding 300 mu LDNA extracting solution into the precipitate, uniformly mixing, incubating at 56 ℃ for 30min, carrying out water bath at 90-100 ℃ for 8 min-10 min, centrifuging at 4 ℃ and 12000rpm for 2min, and taking supernatant as DNA.

When the sample is milk powder, the sample pretreatment method comprises the following steps: dissolving 10g of milk powder in 90mL of sterile peptone water solution (BPW), mixing uniformly, centrifuging at 4 ℃ and 5000rpm for 5min, discarding supernatant, and wiping residual milk fat on the upper layer and the tube wall with a sterilized cotton swab; the pellet was re-solubilized in peptone water solution for a second centrifugation, the above procedure was repeated, and BPW was added for re-solubilization and for DNA extraction.

S2, establishing LAMP system of Cronobacter sakazakii by taking sample DNA obtained from S1 as template, and adding HNB dye for amplification

The LAMP system of cronobacter sakazakii has a total volume of 25 mu L and comprises the following components: primer set, dNTP, Mg described in example 1²⁺10 × ThermoPol Buffer, Bst 2.0WarmStart DNA polymerase and template DNA; the concentration ratio of the outer primer to the inner primer in the primer group is 1:8, the final concentration of dNTP is 1.8 mM-2.2 mM, and Mg²⁺The final concentration is 3-5 mM, the addition amount of 10 × ThermoPol Buffer is 2.5 μ L, the initial concentration of Bst 2.0WarmStart DNA polymerase is 8000U/mL, the addition amount is 0.8 μ L, and the addition amount of template DNA is 2 μ L.

Adding HNB dye into the LAMP system to ensure that the final concentration is 120-180 mu M, and carrying out isothermal amplification. The amplification reaction temperature is 62-64 ℃, and the reaction time is 30-60 min.

(1) Optimization of LAMP reaction system and reaction conditions

Optimization of concentration ratio of outer primer to inner primer

The amount of the inner primer added was adjusted so that the final concentration ratios of the outer primer to the inner primer were 1:2, 1:4, 1:6, 1:8, 1:10 and 1:12, respectively. Preparing a corresponding LAMP system according to the proportion, amplifying for 1h at 65 ℃, and then heating for 5min at 80 ℃ to achieve the purpose of terminating the reaction. 5 mu L of reaction product is detected by using 2% AGE, and the condition that the electrophoretic band is brightest under ultraviolet light is the optimal primer concentration ratio of the reaction.

As shown in FIG. 1, LAMP amplification reaction can occur when the concentration ratio of the outer primers to the inner primers is 1: 2-1: 12, the concentration of the fixed outer primers in the experiment is increased, the electrophoresis band becomes bright and dark first along with the increase of the concentration of the inner primers, the electrophoresis band is brightest and clear when the concentration ratio is 1:8, the amplification effect is best, and the concentration ratio of the outer primers to the inner primers is finally determined to be 1: 8.

② optimization of dNTP concentration

The amounts of dNTPs added were adjusted so that the final concentrations were 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 and 2.4mM, respectively. Preparing a corresponding LAMP system according to the proportion, amplifying for 1h at 65 ℃, and then heating for 5min at 80 ℃ to terminate the reaction. 5 μ L of the reaction product was detected with 2% AGE, and the brightest electrophoretic band under UV light was the optimal dNTP concentration for the reaction.

As shown in FIG. 2, the amplification efficiency was not achieved at a dNTP concentration of 1.2mM, and the electrophoretic bands became brighter and darker as the dNTP concentration increased, and the final concentration of dNTP was determined to be 1.8 mM-2.2 mM.

(iii) optimization of magnesium ion concentration

Regulating Mg²⁺The amounts of (A) were added so that the final concentrations were 1, 2, 3, 4, 5 and 6mM, respectively. Preparing a corresponding LAMP system according to the proportion, amplifying for 1h at 65 ℃, and then heating for 5min at 80 ℃ to terminate the reaction. 5 mul of the reaction product was detected with 2% AGE, the brightest electrophoretic band under UV light being the optimal concentration of magnesium ions for the reaction.

The results are shown in FIG. 3, Mg²⁺The LAMP amplification reaction can be carried out when the concentration is between 1mM and 6mM, the electrophoresis bands are lightened and then darkened along with the increase of the concentration of magnesium ions, and finally Mg is determined²⁺Has a final concentration of Mg²⁺The final concentration is 3-5 mM.

Bst 2.0 optimization of the amount of addition of the WarmStart DNA polymerase

The amounts of the enzymes added were adjusted to 0.6, 0.7, 0.8, 0.9, 1.0, and 1.1. mu.L, respectively. Preparing a corresponding LAMP system according to the proportion, amplifying for 1h at 65 ℃, and then heating for 5min at 80 ℃ to terminate the reaction. 5 μ L of the reaction product was detected by 2% AGE, and the condition of the brightest electrophoretic band under UV light was the optimum amount of enzyme added.

As shown in FIG. 4, the LAMP amplification reaction occurred in all cases when the amount of DNA polymerase was between 0.6. mu.L and 1.1. mu.L, the electrophoretic bands became bright and dark first with increasing enzyme amount, the electrophoretic bands became brightest and clear at an enzyme concentration of 0.8. mu.L, indicating that the amplification effect was the best, and the amount of Bst 2.0DNA polymerase was finally determined to be 0.8. mu.L.

Optimization of reaction temperature

The reaction temperature was adjusted to 60, 61, 62, 63, 64, 65, 66 ℃ respectively, the amplification time was 1h, and then the reaction was terminated by heating at 80 ℃ for 5 min. 5 μ L of the reaction product was detected by 2% AGE, and the condition of the brightest electrophoretic band under UV light was the optimal reaction temperature.

As shown in FIG. 5, LAMP amplification reaction can occur at a reaction temperature of 60-66 deg.C, and electrophoresis bands become bright and dark with the increase of the reaction temperature, and the reaction temperature is finally determined to be 62-64 deg.C.

Optimization of amplification time

Amplifying at 65 deg.C for 30, 45, 60, 75, 90min, and heating at 80 deg.C for 5min to terminate the reaction. 5 μ L of the reaction product was detected with 2% AGE and the shortest amplification time with stable electrophoretic band brightness under UV light was chosen.

As shown in FIG. 6, LAMP amplification reaction can occur in the amplification time of 30min or more, the electrophoresis bands become bright and dark with the increase of the reaction temperature, and the reaction time is finally determined to be 30-60 min.

Determination of LAMP System

The optimal amplification system is established by optimizing each component and reaction condition in the LAMP system as shown in Table 2.

Table 2 optimal LAMP amplification System for detecting Cronobacter sakazakii

Preferably, the reaction is terminated by amplification at a constant temperature of 63 ℃ for 1h, followed by treatment at 80 ℃ for 5min to inactivate the enzyme.

(2) Optimization of HNB dye addition

The HNB powder is diluted to 1.5mM by deionized water to be used as mother liquor for subpackaging, and is stored at the temperature of minus 20 ℃ in a dark place for standby. Then, the concentration of the HNB dye in the LAMP system was optimized, the addition amount of the HNB dye mother liquor was adjusted to the final concentrations of 90, 120, 150 and 180. mu.M, and LAMP amplification reaction was carried out with 2. mu.L of ddH2O instead of DNA as negative control. Selecting the optimal concentration of HNB according to the condition that the positive result shows sky blue and the negative result shows violet blue.

As shown in FIG. 7, when the concentration of HNB was not less than 120. mu.M, the positive reaction system appeared bluish and the negative was purple, and the final concentration of HNB in the system was finally determined to be 120. mu.M-180. mu.M.

(3) LAMP-HNB detection method sensitivity determination

Activation and culture of strains

Adding frozen bacteria liquid into a 20ml LTSB liquid culture medium according to the proportion of 2-3%, culturing for 8h under the conditions of 37 ℃ and 200r/min, and repeatedly activating the bacteria liquid for the second time. And then carrying out three-zone streaking on the TSA solid culture medium, culturing for 14-16 h at 37 ℃ for purification, selecting a single colony in the purified TSA solid culture medium to be cultured in 20mL of TSB liquid culture medium for 8h at 37 ℃ in a shaking table at 200r/min to obtain a strain at the end of a logarithmic phase, and extracting DNA from part of bacterial liquid for subsequent experiments.

Colony counting method

Counting was performed by the spread plate method. The bacterial culture solution was diluted with sterilized 0.85% physiological saline and serially diluted 10-fold in gradient. Then respectively taking the dilution times as 10^-4、10^-5、10^-6The plate coating was performed in 3 gradients, three in parallel per gradient. And (3) placing the coated plate at 37 ℃ for culturing for 12-18 h, and enabling the plate to grow single colonies and counting. The colony range selected by counting the plates is 30-300, and the viable bacteria concentration (CFU/mL) of the original bacteria liquid is obtained by multiplying the average number of the colonies of the three plates by the dilution times and then multiplying by 10.

Amplification of DNA

The extracted DNA was amplified according to the established LAMP-HNB system, and the DNA of Cronobacter sakazakii was amplified according to the PCR reaction system shown in Table 3.

TABLE 3PCR reaction System

Through systematic optimization experiments, the optimal reaction conditions are obtained as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 30s, for 30 cycles, and final extension at 72 ℃ for 5 min. mu.L of the reaction product was detected by 1% Agarose Gel Electrophoresis (AGE), and the band was observed by UV gel imaging.

(ii) detection sensitivity of pure culture

Selecting Cronobacter sakazakii ATCC 29544 as a standard strain to be cultured, culturing for 8h at 37 ℃ and 200r/min in 20mL of TSB culture medium to reach a growth log phase, performing 10-fold gradient dilution on the bacterial liquid by using 0.85% physiological saline, coating and counting, determining the viable bacteria concentration of the bacterial liquid, and ensuring the concentration range of target bacteria to be 10⁶～10^-1And (3) extracting DNA of bacterial liquids with different concentrations between CFU/mL to perform LAMP-HNB detection, and simultaneously performing comparative analysis with the traditional PCR method.

The detection results are as follows:

the results of the detection of sensitivity were as shown in FIG. 8, and the concentration of Cronobacter sakazakii was determined to be in the range of 4.5X 10 by plate counting⁶CFU/mL to 4.5X 10^-1CFU/mL. In the case where the control group showed negative, when the concentration of the pure culture was 4.5X 10⁶The detection results are positive when CFU/mL is between 4.5X 100CFU/mL, and are 10^-1CFU/mL concentration was negative. Meanwhile, the concentration range of the PCR method in the cronobacter sakazakii is 4.5 multiplied by 10⁶CFU/mL to 4.5X 10²The result of the electrophoresis detection is positive when the concentration is CFU/mL, and the result is negative when the concentration is lower than the CFU/mL. Therefore, the LAMP-HNB method has a detection sensitivity of 4.5X 100CFU/mL and the PCR-AGE method has a sensitivity of 4.5X 10²CFU/mL, the detection sensitivity of the visualization method established by the invention is 100 times that of the traditional PCR method.

② the detection sensitivity of Cronobacter sakazakii in the infant formula

The purchased PIF was tested according to the conventional test method suggested and recommended by the FDA to confirm that the powdered milk contains no cronobacter sakazakii at all. Cronobacter sakazakii ATCC 29544 was then artificially contaminated into sterile PIF: dissolving 10g of milk powder in 90mL of sterile peptone water solution (BPW), mixing uniformly, performing pretreatment steps such as centrifugation and heavy suspension, and adding pure culture bacteria solutions with different dilutions to obtain the final concentration range of Cronobacter sakazakii of 10⁶～10^-1Artificially contaminated PIF samples between CFU/g. Extracting the genomic DNA of Cronobacter sakazakii in the milk sample to carry out LAMP-HNB detection, and simultaneously comparing with the traditional PCR method.

The detection results are as follows:

the results of detection of the sensitivity to Cronobacter sakazakii in PIF are shown in FIG. 9, and the concentration of Cronobacter sakazakii in the artificially contaminated milk was determined to be in the range of 5.7X 10 by plate counting⁶CFU/g to 5.7 x 10^-1CFU/g. When the concentration of the target bacteria is 5.7 multiplied by 10⁶The HNB dye-carrying system shows sky blue with positive results when CFU/g is 5.7 multiplied by 10CFU/g, and each control group shows negative results when CFU/g is 5.7 multiplied by 10 CFU/g. And at 5.7X 100CFU/g and 5.7X 10^-1CFU/g concentration was negative. At the same time, PCR method is performed in sakaThe concentration range of Cronobacter kazakii is 5.7 × 10⁶CFU/g to 5.7 x 10³The band appeared in the electrophoresis at CFU/g is a positive result, and the band appeared below the concentration is a negative result. Therefore, the LAMP-HNB method has the detection sensitivity of 5.7 multiplied by 10CFU/g for Cronobacter sakazakii in milk powder, and the PCR-AGE method has the sensitivity of 5.7 multiplied by 10CFU/g³CFU/g, the detection sensitivity of the visualization method established by the invention is 100 times that of the traditional PCR method.

(4) Specificity of LAMP-HNB detection method

By applying the constructed LAMP-HNB visual detection method, specificity verification is carried out on 8 Cronobacter sakazakii strains and 14 Cronobacter sakazakii strains, the color change of the LAMP-HNB system is observed, and the specificity of the detection method is judged. Three parallel runs were performed for each strain.

Results as shown in table 4, all of the 8 cronobacters sakazakii were positive in the visual detection results, and 14 cronobacters sakazakii were negative in the amplification results. Therefore, the LAMP-HNB visual detection method based on the novel nucleic acid target point is proved to have specificity to the Cronobacter sakazakii.

Note: "+" represents positive; "-" represents negativity

(5) Practicability evaluation of LAMP-HNB visual detection method

50 parts of PIF were purchased from retail markets nationwide and determined to be free of Clonobacter sakazakii according to the national Standard microbiological examination method (GB 4789.40-2016). And then selecting 30 parts of milk powder to carry out artificial pollution on Cronobacter sakazakii and using the milk powder as a positive sample, controlling the concentration of a bacterial liquid to be 101CFU/mL, using the other 20 parts of milk powder as a negative sample, respectively amplifying by using the constructed LAMP system, detecting by using an LAMP-HNB visualization method, and judging the coincidence rate and the practicability of the milk powder according to results.

The result of the utility evaluation:

the visual detection method constructed by the invention is used for detecting 30 parts of artificially polluted milk, and the amplification results are positive; and detecting 20 parts of normal pollution-free milk powder, wherein amplification results are negative. Therefore, the LAMP-HNB visualization method for detecting cronobacter sakazakii established in the research has the advantages that the coincidence rate of the detected milk powder is 100%, and the practicability is high.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> northeast university of agriculture

<120> novel target, primer set and detection method for cronobacter sakazakii detection

<160> 5

<170> PatentIn version 3.5

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<211> 211

<212> DNA

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<400> 1

atctcgctgc ccgctatcgc tataacacgg tgaatgatgc ccatagcgac agaaagctcg 60

acagcgatga gtatacccgt cgtaaatcgc atcagtttga tatctggatt gcctataata 120

tcgggcgctt cggtatgtcc tggaaccctc gcttccgtta ccaggatggc gtcgatcagg 180

gaaccgggag agacacgtac tgggagcata c 211

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<212> DNA

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atctcgctgc ccgctatc 18

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<212> DNA

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gtatgctccc agtacgtgtc 20

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<211> 40

<212> DNA

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actgatgcga tttacgacgg gtcacggtga atgatgccca 40

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<212> DNA

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<400> 5

ggcgcttcgg tatgtcctgg tctcccggtt ccctgatc 38

Claims

1. The application of the nucleotide sequence in detecting cronobacter sakazakii is characterized in that the nucleotide sequence is shown as SEQ ID No. 1.

2. The primer group for detecting cronobacter sakazakii, which is designed based on the nucleotide sequence of claim 1, and specifically comprises the following primers:

outer primer F3: 5'-ATCTCGCTGCCCGCTATC-3', respectively;

outer primer B3: 5'-GTATGCTCCCAGTACGTGTC-3', respectively;

inner primer FIP: 5'-ACTGATGCGATTTACGACGGGTCACGGTGAATGATGCCCA-3', respectively;

3. A method for visually detecting Cronobacter sakazakii is characterized by comprising the following steps:

4. The method according to claim 3, wherein the DNA extract solution of S1 comprises Chelex-1005g beads, NP 401mL, and TE solution 19mL, and has a pH of 8.0 to 9.0; the TE solution was prepared from 10mM Tris-HCl and 1mM EDTA.

5. The method according to claim 3, wherein the DNA extraction method of S1 is as follows: taking 300 mu L-1 mL of sample to be detected in a 2mL EP tube, centrifuging at 4 ℃ and 12000rpm for 2min, discarding supernatant, adding 300 mu LDNA extracting solution into the precipitate, uniformly mixing, incubating at 56 ℃ for 30min, carrying out water bath at 90-100 ℃ for 8 min-10 min, centrifuging at 4 ℃ and 12000rpm for 2min, and taking supernatant as DNA.

6. The method of claim 3, wherein S2 the LAMP system of Cronobacter sakazakii has a total volume of 25 μ L and comprises the following components: the primer set, dNTP, Mg according to claim 2²⁺10 × ThermoPol Buffer, Bst 2.0WarmStart DNA polymerase and template DNA; the concentration ratio of the outer primer to the inner primer in the primer group is 1:8, the final concentration of dNTP is 1.8 mM-2.2 mM, and Mg²⁺The final concentration is 3-5 mM, the addition amount of 10 × ThermoPol Buffer is 2.5 μ L, the initial concentration of Bst 2.0WarmStart DNA polymerase is 8000U/mL, the addition amount is 0.8 μ L, and the addition amount of template DNA is 2 μ L.

7. The method of claim 3, wherein the final concentration of the HNB dye of S2 is 120 μ M to 180 μ M.

8. The method according to claim 3, wherein the temperature of the amplification reaction of S2 is 62 ℃ to 64 ℃.

9. The method according to claim 3, wherein the time for the amplification reaction of S2 is 30-60 min.

10. The method according to claim 3, wherein when the sample of S1 is milk powder, the sample pretreatment method comprises: dissolving 10g of milk powder in 90mL of sterile peptone water solution, mixing uniformly, centrifuging at 4 ℃ and 5000rpm for 5min, and discarding the supernatant; the pellet was re-solubilized in peptone water solution for a second centrifugation, added with BPW re-solubilization and used for DNA extraction.