Primer probe combination, digital PCR (polymerase chain reaction) detection kit and method for absolute quantitative detection of cronobacter in food

Abstract

The invention relates to a primer probe combination, a digital PCR (Polymerase Chain Reaction) detection kit and a method for absolute quantitative detection of cronobacter in food. The primer probe combination comprises a recN-F upstream primer, a recN-R downstream primer and a recN-P probe, the nucleotide sequence of the recN-F upstream primer is as shown in SEQ ID NO.1, the nucleotide sequence of the recN-R downstream primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the recN-P probe is as shown in SEQ ID NO.3. The invention further discloses a kit for detecting the protein content of the protein. Components participating in amplification are divided into millions of PCR reactions which obey Poisson distribution, then each reaction is subjected to PCR amplification, then the positive and negative properties of reaction results are judged by reading fluorescence signals, and finally the copy number and concentration of target molecules are calculated according to the judgment result according to the Poisson distribution principle, so that absolute quantification is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological pathogen detection, and in particular to a primer-probe combination, a digital PCR detection kit and a method for absolute quantitative detection of Cronobacter in food. Background Art

[0002] Cronobacter is a facultative anaerobic, Gram-negative, opportunistic pathogen that possesses periodic flagella, is motile, and lacks spores. It is a significant foodborne pathogen and is extremely dangerous to infants and young children, particularly premature infants, those with low body weight, and those with compromised immune systems. Infection can cause meningitis, sepsis, and enterocolitis, among other illnesses. Numerous cases of infection have been reported worldwide in recent years. In addition to neonatal infections, the bacterium can, under certain conditions, infect the elderly and immunocompromised adults, leading to bacteremia, osteomyelitis, and other illnesses.

[0003] Cronobacter is widely distributed in nature and has been detected in water, soil, and sewage. It has also been detected in a variety of foods, such as cheese products, meat, rice and other grains, vegetables, fermented bread, poultry, milk, pasta, cereals, etc. However, the contamination of Cronobacter with different types of food varies greatly, and the detection rate of Cronobacter in cereals is relatively high. In 2022, the International Commission on Microbiological Specifications for Foods (ICMSF) listed Enterobacter sakazakii (Cronobacter) as "a pathogen that seriously endangers the lives of specific populations and causes long-term chronic substantial sequelae." In 2004, the Food and Agriculture Organization of the United Nations and the World Health Organization listed Enterobacter sakazakii (Cronobacter) and Salmonella as Category A pathogens in infant formula powder after risk assessment. Therefore, conducting risk assessment of Cronobacter in food is of great reference significance for controlling the safety risks brought by it and taking prevention and control measures. In addition, risk assessment is an important scientific means to develop quality control systems, set food safety goals and ultimately achieve food safety risk control.

[0004] In 1998, the Codex Alimentarius Commission (CAC) established guidelines for risk assessment. Microbial risk assessment is divided into four steps: hazard identification, exposure assessment, hazard characterization, and risk characterization. Hazard identification is the first step in the microbial risk assessment process. Its core component is identifying the type of microbial hazard in food and determining the potential health consequences and adverse reactions that could result from consuming food contaminated with the pathogenic microorganism. Currently, the maximum likelihood number (MPN) method (GB4789.10-2024) is a common hazard identification technique used in Cronobacter risk assessment. This method applies probability theory to estimate bacterial concentrations. This method also ultimately relies on bacterial isolation and identification on plates, which is cumbersome and time-consuming, and does not conform to the "rapid, high-throughput" trend of advanced detection methods. While DNA-based PCR amplification methods offer the advantages of efficiency and speed, they cannot quantify bacteria, making their results difficult to use for quantitative microbial risk assessment. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a primer probe combination, a digital PCR detection kit and a method for absolute quantitative detection of Cronobacter in food, so as to solve the above problems.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] In order to achieve the above object, the first aspect of the present invention provides a primer-probe combination for absolute quantitative detection of Cronobacter in food, characterized in that it includes a recN-F upstream primer, a recN-R downstream primer and a recN-P probe;

[0008] The nucleotide sequence of the recN-F upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the recN-R downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the recN-P probe is shown in SEQ ID NO.3.

[0009] A second aspect of the present invention provides a digital PCR detection kit for absolute quantitative detection of Cronobacter in food, comprising the above-mentioned primer-probe combination.

[0010] As a further technical solution of the present invention: the kit also includes sterile water, PCR reaction solution, a blank control substance, a positive control substance and a negative control substance.

[0011] As a further technical solution of the present invention: the blank control is RNase-free ultrapure water, the positive control is the DNA of the recombinant plasmid pMD19-T-recN or the DNA of the target bacteria, and the plasmid negative control is the DNA of pMD19-T or bacterial DNA without specific genes.

[0012] A third aspect of the present invention provides a method for absolute quantitative detection of Cronobacter in food using the above-mentioned kit, comprising the following steps:

[0013] Step 1. Design and synthesize primers and probes: Primers and probes were designed based on the analysis and alignment of five different Cronobacter gene sequences published on the NCBI official website. The nucleotide sequence of the recN-F upstream primer is shown in SEQ ID NO. 1, the nucleotide sequence of the recN-R downstream primer is shown in SEQ ID NO. 2, and the nucleotide sequence of the recN-P probe is shown in SEQ ID NO. 3.

[0014] Step 2. Prepare control substances: Prepare DNA of plasmid pMD19-T or bacterial DNA without specific genes as negative control substances, prepare DNA of plasmid pMD19-T-recN or DNA of target bacteria as positive control substances, and use RNase-free ultrapure water as blank control substance;

[0015] Step 3, droplet generation: Extract the DNA of the sample to be tested, use the primers and probes designed in step 1, and use the DNA of the sample to be tested as a template in the sample preparation area. Use a sample preparation instrument to generate droplets. After the droplet generation is completed, immediately perform the PCR amplification reaction, or place it in an environment of 2-8°C and start the PCR amplification reaction within 1 hour;

[0016] Step 4: Result detection: Use an analyzer to detect the droplets and determine whether the sample is positive or negative.

[0017] As a further technical solution of the present invention: in step 3, the method for preparing the sample DNA comprises the following steps: taking a 25 g / mL sample by aseptic operation, placing it in a sterile homogenizing cup containing 225 mL of BPW, and homogenizing it at 8000 rpm to 10000 rpm for 1 to 2 minutes, or placing it in a sterile homogenizing bag containing 225 mL of BPW and beating it with a slapping homogenizer for 1 to 2 minutes;

[0018] Pipette 10 mL of the sample solution and transfer it to a centrifuge tube. Centrifuge at 4000 rpm for 15 min at room temperature and discard the supernatant.

[0019] The precipitate was resuspended with sterile water, transferred to a 1.5 mL EP tube, centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the DNA of the precipitated sample was extracted.

[0020] As a further technical solution of the present invention: the amplification reaction conditions of step 3 are: pre-denaturation at 95° C. for 10 min; denaturation at 94° C. for 30 s; annealing at 60° C. for 1 min; a total of 40 cycles.

[0021] As a further technical solution of the present invention: the effectiveness of the detection in step 4 is determined as follows:

[0022] The following quality control requirements must be met: the blank control is negative; the negative control is negative; the positive control is positive; there is a clear boundary between negative droplets and positive droplets, and the number of positive droplets must not be too close to the total number of droplets.

[0023] As a further technical solution of the present invention: the judgment standard of the detection result of step 4 is: if the total number of droplets in the reaction well of the 30 μL reaction system is less than 30,000, or the total number of droplets in the reaction well of the 20 μL reaction system is less than 20,000, it indicates that the microdroplet generation / detection in the reaction well is not ideal, and the sample to be tested needs to be retested.

[0024] In addition, the present invention also provides the use of the above-mentioned primer-probe combination in the preparation of a product for detecting Cronobacter in food, and the product can be a kit, a PCR detection reagent, a detection chip, etc.

[0025] In summary, the present invention includes at least one of the following beneficial technical effects:

[0026] 1. Compared with the existing technology, the advantages and positive effects of the present invention are: a primer-probe combination, a digital PCR detection kit and a method for the absolute quantitative detection of Cronobacter in food are proposed. The digital PCR technology can achieve rapid detection of Cronobacter in food and absolute quantification of it. Droplet digital PCR (ddPCR) is a technology for absolute quantification of nucleic acid molecules based on traditional PCR. It divides the components involved in amplification into millions of PCR reactions, so that each reaction contains as much template molecule as possible. Each reaction is then PCR amplified, and the positive and negative reaction results are judged by reading the fluorescence signal. Finally, the judgment result is used to estimate the copy number and concentration of the target molecule according to the principle of Poisson distribution, thereby achieving absolute quantification.

[0027] 2. Compared with the existing technology, the advantages and positive effects of the present invention are: this patent can realize the absolute quantification of Cronobacter in food while realizing the rapid detection. At present, the hazard identification technology in Cronobacter risk assessment often uses the Maximum Likelihood Number method (Most Probable Number, MPN) (GB4789.10-2024), which is a method of estimating bacterial concentration by applying probability theory. Moreover, this method ultimately obtains results by separating and identifying bacteria on plates. The operation is cumbersome and time-consuming, which does not conform to the trend of "fast, high-throughput, and accurate" of advanced detection methods. This patent can make up for the shortcomings of the existing technology and realize "fast, high-throughput, and accurate" absolute quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The colony concentration in the present invention is 1.67×10 6 CFU / mL test results chart.

[0029] Figure 2 The colony concentration in the present invention is 1.81×10 5 CFU / mL test results chart.

[0030] Figure 3 The colony concentration in the present invention is 1.34×10 4 CFU / mL test results chart.

[0031] Figure 4 The colony concentration in the present invention is 1.34×10 4 CFU / mL test results chart.

[0032] Figure 5 This is a graph showing the detection results of the colony concentration of 330 CFU / mL in the present invention.

[0033] Figure 6 Schematic diagram showing the comparison of the results of detecting Cronobacter in bacterial liquid by ddPCR and plate count method in the present invention.

[0034] Figure 7 Schematic diagram showing the comparison of the results of ddPCR and plate count method in the present invention for detecting Cronobacter in spiked milk powder. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application; it is obvious that the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0036] Example 1:

[0037] 1. Materials and Methods

[0038] Specific genes identified by Cronobacter: recN gene encoding recombination and repair proteins. The sequence reference of the gene comes from 5 different Cronobacter species:

[0039] NCBI official website Cronobacter dublinensis subsp.dublinensis LMG 23823 (NZ_CP012266.1), Cronobacter muytjensii ATCC 51329 (NZ_CP012268.1), Cronobacter malonaticus LMG 23826 (NZ_CP013940.1), Cronobacter turicensis LMG23827(NZ_CP165997.1), Cronobacter sakazakii CS-931(NZ_CP027107.1).

[0040] 1.1 Primer and probe sequences are as follows:

[0041] recN-F upstream primer: 5'-CAATCTGCAAAGCCAGCTCTA-3', SEQ ID NO.1

[0042] recN-R downstream primer: 5'-GCGGCCTCTTCAAGCATATT-3', SEQ ID NO.2

[0043] recN-P probe: 5'-FAM-ATCTCGTCGGCATGGATGACAAACTC-BHQ1-3', SEQ ID NO.3

[0044] 1.2ddPCR detection kit

[0045] The system is based on 30 μL, including 7.5 μL of reaction premix 4×Probe dPCR Unimix (with UNG), 0.9 μL of upstream and downstream primers (final concentration 300 nmol / L), 0.45 μL of probe (final concentration 150 nmol / L), and 1.0 μL of DNA template. Double-distilled water is used to make up the total volume to 30 μL.

[0046] 1.3ddPCR detection method:

[0047] Pre-denaturation at 95°C for 10 min

[0048] Denature at 94°C for 30 seconds

[0049] Annealing at 60°C for 1 min

[0050] 40 cycles.

[0051] 1.4 ddPCR detection of Cronobacter in food

[0052] 1.4.1 Extract genomic DNA from food samples;

[0053] 1.4.2 Amplify using the above primer-probe combination and ddPCR kit according to the ddPCR detection method;

[0054] 1.4.3 Result determination:

[0055] The droplet analyzer detects each droplet after PCR amplification one by one, interpreting droplets with a fluorescent signal as 1 and droplets without a fluorescent signal as 0. The starting copy number or concentration of the gene to be tested is derived based on the Poisson distribution of the number of positive droplets and the total number of droplets. Therefore, a 30μL reaction system must generate at least 30,000 droplets, and the number of positive droplets cannot be too close to the total number of droplets, requiring a clear boundary between negative and positive droplets. A 20μL reaction system must generate at least 20,000 droplets, and the number of positive droplets cannot be too close to the total number of droplets, requiring a clear boundary between negative and positive droplets.

[0056] Comparative analysis of 1.6ddPCR detection results and plate count results (Supplementary Data)

[0057] The plate count result of fresh culture solution was 1.58×10 7 CFU / mL, and then conduct gradient dilution, and then extract DNA from the bacterial suspension of different dilutions, and perform ddPCR detection, and at the same time perform plate count detection on the bacterial suspension of each dilution, the results are as follows Figure 1-Figure 5 shown.

[0058] Table 1 Analysis of ddPCR and plate count results for detection of Cronobacter

[0059]

[0060] Refer to Table 1 and Figure 6 The results showed that the probe primer combination in this patent was used to detect Cronobacter by ddPCR, and the results (copy number / copies converted to colony count / CFU / mL) were consistent with the plate count results (Log 10 CFU / mL) showed a good linear relationship: R 2=0.9982. Furthermore, for bacterial extracts of different concentrations, the relative standard deviations of the detection results of the two methods ranged from 0.14 to 2.5, further confirming the reliability of ddPCR for quantitative detection of Cronobacter.

[0061] 1.5ddPCR amplification specificity

[0062] A total of 13 standard strains and various food isolates from Shanghai's food safety risk monitoring and 5 other non-Cronobacter (Salmonella, Staphylococcus aureus, diarrheagenic Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca) preserved in this laboratory were selected for detection using the probe primers and ddPCR detection kit and method invented in this patent. The results showed that the amplification results of the 13 food-isolated Cronobacter all showed specific amplification, while the results of the 5 non-Cronobacter were negative. Referring to Table 2, the specificity of the ddPCR detection results developed in this patent was further confirmed.

[0063] Table 2 Information on strains used in this patent and ddPCR test results

[0064]

[0065] 1.6 ddPCR detection sensitivity of Cronobacter in artificially contaminated milk powder and comparison of the results with the corresponding bacterial suspension concentration

[0066] 1.6.1 ddPCR detection sensitivity, compared with plate count results:

[0067] The original bacterial plate count result was 1.87×10 8 CFU / mL. Perform a serial dilution of the original bacterial suspension. Weigh 10 g of milk powder and add 90 mL of sterile water. Add the serially diluted bacterial suspension at a ratio of 1:10 to prepare spiked samples. Take 1 mL of spiked samples of different concentrations to extract DNA for ddPCR analysis. Simultaneously, take 1 mL of spiked sample for plate count.

[0068] The results showed that the limit of ddPCR detection was 151 CFU / mL, and the ddPCR detection results were well correlated with the plate count results. 2 =0.9963, the result is as follows Figure 7 and as shown in Table 3. At the same time, the relative deviation of the logarithmic values of the ddPCR detection results and the plate count results was analyzed. The detection results of the spiked samples with six different bacterial solution dilutions showed that the relative standard deviation of the logarithmic values of the results of the two different methods was 5.26% at most and 0.21% at least, further confirming the effectiveness of ddPCR in quantitative detection of Cronobacter.

[0069] Table 3 Analysis of ddPCR and plate count results for detecting Cronobacter in spiked milk powder

[0070]

[0071] 2. Detailed operation method for developing a kit to detect Cronobacter in food:

[0072] 2.1 Pretreatment of food samples

[0073] 2.1.1 Aseptic operation: Take 25 g (mL) of sample and place it in a sterile homogenizing cup containing 225 mL of BPW. Homogenize at 8000-10000 r / min for 1-2 min. Alternatively, place it in a sterile homogenizing bag containing 225 mL of BPW and beat it with a slapping homogenizer for 1-2 min.

[0074] 2.1.2 Transfer 10 mL of the sample solution to a centrifuge tube, centrifuge at 4000 rpm for 15 min at room temperature, and discard the supernatant.

[0075] 2.1.3 Resuspend the pellet in sterile water, transfer to a 1.5 mL EP tube, centrifuge at 8000 rpm for 3 minutes, discard the supernatant, and extract the DNA from the pellet. Note that chromosomes should be extracted immediately.

[0076] 2.2 DNA extraction method

[0077] DNA extraction and purification were performed using a commercial DNA extraction kit according to the kit's instructions.

[0078] 2.3 Quality Control

[0079] 2.3.1 Blank control

[0080] The blank control was RNase-free ultrapure water.

[0081] 2.3.2 Negative Control

[0082] Negative controls were DNA from plasmid pMD19-T or bacterial DNA without the specific gene.

[0083] 2.3.3 Positive Control

[0084] The positive control is the DNA of the recombinant plasmid pMD19-T-recN or the DNA of the target bacteria.

[0085] 2.4 Sample testing

[0086] 2.4.1 Reaction system preparation

[0087] Table 4 Reaction system (30 μL)

[0088]

[0089] Table 5 Reaction system (20 μL)

[0090]

[0091] 2.4.2 Droplet generation

[0092] In the sample preparation area, droplets are generated using a sample preparation instrument. After droplet generation is complete, PCR amplification is performed immediately or within 1 hour of placement at 2-8°C.

[0093] 2.4.3 Digital PCR Reaction System

[0094] Table 6 Reaction system

[0095]

[0096] 2.4.4 Testing of blank control, negative control, and positive control

[0097] The blank control was RNase-free ultrapure water.

[0098] Negative control: Prepare the reaction system according to 2.4.1, replacing the sample DNA extract with negative control DNA.

[0099] Positive control: Prepare the reaction system according to 2.4.1, replacing the sample DNA extract with positive control DNA.

[0100] The digital PCR reaction system is the same as 2.4.3.

[0101] 2.4.5 Droplet Detection

[0102] The droplets are detected using an analyzer.

[0103] 3. Results and Report

[0104] 3.1 Determination of test effectiveness

[0105] 3.1.1 The following quality control requirements must be met: blank control is negative; negative control is negative; positive control is positive.

[0106] 3.1.2 There should be a clear boundary between negative droplets and positive droplets, and the number of positive droplets should not be too close to the total number of droplets.

[0107] 3.2 Result determination

[0108] 3.2.1 If the total number of droplets in a reaction well for a 30 μL reaction system is less than 30,000, or the total number of droplets in a reaction well for a 20 μL reaction system is less than 20,000, this indicates that droplet generation / detection in that reaction well was unsatisfactory and the sample should be retested.

[0109] 3.2.2 Report

[0110] The Cronobacter content was calculated according to the following formula:

[0111]

[0112] Note: * refers to the volume of the reaction system. If the volume of the reaction system is 20 μL, then this is 20 μL.

[0113] The embodiments of this specific implementation method are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A primer-probe combination for absolute quantitative detection of Cronobacter in food, characterized in that: Including recN-F upstream primer, recN-R downstream primer and recN-P probe; The nucleotide sequence of the recN-F upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the recN-R downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the recN-P probe is shown in SEQ ID NO.

3.

2. Use of the primer-probe combination for absolute quantitative detection of Cronobacter in food according to claim 1 in preparing a product for detecting Cronobacter in food.

3. A digital PCR detection kit for absolute quantitative detection of Cronobacter in food, characterized in that: The method comprises the primer-probe combination according to claim 1.

4. A digital PCR detection kit for absolute quantitative detection of Cronobacter in food according to claim 3, characterized in that: It also includes sterile water, PCR reaction solution, blank control, positive control and negative control.

5. A digital PCR detection kit for absolute quantitative detection of Cronobacter in food according to claim 4, characterized in that: The blank control substance is RNase-free ultrapure water, the positive control substance is the DNA of the recombinant plasmid pMD19-T-recN or the DNA of the target bacteria, and the plasmid negative control substance is the DNA of pMD19-T or bacterial DNA without specific genes.

6. A method for absolute quantitative detection of Cronobacter in food, characterized in that: Using the kit according to any one of claims 3 to 5, the detection method comprises the following steps: Step 1. Design and synthesize primers and probes: Primers and probes were designed based on the analysis and alignment of five different Cronobacter gene sequences published on the NCBI official website. The nucleotide sequence of the recN-F upstream primer is shown in SEQ ID NO. 1, the nucleotide sequence of the recN-R downstream primer is shown in SEQ ID NO. 2, and the nucleotide sequence of the recN-P probe is shown in SEQ ID NO.

3. Step 2. Prepare control substances: Prepare DNA of plasmid pMD19-T or bacterial DNA without specific genes as negative control substances, prepare DNA of plasmid pMD19-T-recN or DNA of target bacteria as positive control substances, and use RNase-free ultrapure water as blank control substance; Step 3, droplet generation: Extract the DNA of the sample to be tested, use the primers and probes designed in step 1, and use the DNA of the sample to be tested as a template in the sample preparation area. Use a sample preparation instrument to generate droplets. After the droplet generation is completed, immediately perform the PCR amplification reaction, or place it in an environment of 2-8°C and start the PCR amplification reaction within 1 hour; Step 4: Result detection: Use an analyzer to detect the droplets and determine whether the sample is positive or negative.

7. A method for absolute quantitative detection of Cronobacter in food according to claim 6, characterized in that: In step 3, the sample DNA preparation method includes the following steps: taking a 25 g / mL sample by aseptic operation, placing it in a sterile homogenizing cup containing 225 mL of BPW, and homogenizing it at 8000 rpm to 10000 rpm for 1 to 2 minutes, or placing it in a sterile homogenizing bag containing 225 mL of BPW and beating it with a slapping homogenizer for 1 to 2 minutes; Pipette 10 mL of the sample solution and transfer it to a centrifuge tube. Centrifuge at 4000 rpm for 15 min at room temperature and discard the supernatant. The precipitate was resuspended with sterile water, transferred to a 1.5 mL EP tube, centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the DNA of the precipitated sample was extracted.

8. A method for absolute quantitative detection of Cronobacter in food according to claim 6, characterized in that: The amplification reaction conditions in step 3 are: pre-denaturation at 95° C. for 10 min; denaturation at 94° C. for 30 s; annealing at 60° C. for 1 min; and 40 cycles in total.

9. A method for absolute quantitative detection of Cronobacter in food according to claim 6, characterized in that: The effectiveness of the detection in step 4 is determined as follows: The following quality control requirements must be met: the blank control is negative; the negative control is negative; the positive control is positive; there is a clear boundary between negative droplets and positive droplets, and the number of positive droplets must not be too close to the total number of droplets.

10. A method for absolute quantitative detection of Cronobacter in food according to claim 6, characterized in that: The judgment criteria for the detection results of step 4 are: if the total number of droplets in the reaction well of the 30 μL reaction system is less than 30,000, or the total number of droplets in the reaction well of the 20 μL reaction system is less than 20,000, it indicates that the microdroplet generation / detection in the reaction well is not ideal and the sample to be tested needs to be retested.

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