CN114457175A - Microdroplet digital PCR method for detecting bifidobacteria in milk powder and application - Google Patents
Microdroplet digital PCR method for detecting bifidobacteria in milk powder and application Download PDFInfo
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Abstract
The invention relates to the technical field of detection, and particularly discloses a microdroplet digital PCR method for detecting bifidobacteria in milk powder and application thereof. The microdroplet digital PCR method for detecting the bifidobacteria in the milk powder comprises a pretreatment step of a sample to be detected, wherein the pretreatment step comprises the following steps of: (1) centrifuging the solution of the product to be detected, and removing the supernatant; (2) carrying out cold and hot alternate treatment; (3) digestion with proteinase K; (4) digesting with lysozyme; (5) digestion was performed with proteinase K and buffer GA. The detection method of the invention has the advantages of high speed, good specificity and high accuracy, and is suitable for quantitative detection of bifidobacteria in milk powder products.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a microdroplet digital PCR method for detecting bifidobacteria in milk powder and application thereof.
Background
The infant formula milk powder is a product prepared by taking milk and milk protein products as main protein sources, adding a proper amount of vitamins, mineral substances and/or other auxiliary materials and producing and processing by a physical method, wherein energy and nutritional ingredients of the product can partially meet the growth needs of infants from birth to 3 years of age, and the product mainly comprises infant formula milk powder and infant formula milk powder for older infants and young infants according to the nutritional needs of the infants at different ages.
The infant formula milk powder is added with a certain amount of probiotics, so that the flora balance of infant intestinal tracts in the 'immune incomplete stage' can be maintained, the dominant position of the probiotics is kept, the immune system of infants is improved, the digestion and absorption of intestines and stomach are promoted, and the normal growth and development of the infants are effectively promoted. As the body functions of infants are not sound, the nutrition community generally considers that the probiotics for supplementing the infants only allow the addition of nonpathogenic L (+) -lactic acid-producing probiotics, mainly comprise bifidobacteria and also comprise certain specific strains of lactobacilli. In order to standardize the use of strains, 13 strains of 11 probiotics which are generally applied to infant food at present mainly comprise bifidobacteria and lactobacilli. Such as Bifidobacterium animalis Bb-12, Bifidobacterium lactis HN019, Bi-07 and Lactobacillus acidophilus NCFM.
The safety of the infant formula milk powder is ensured by the specification of the types of the added probiotics, and the viable count of the probiotic product is a key factor for ensuring the functional characteristics of the infant formula milk powder, and generallyThe number of viable bacteria should not be less than 106CFU/g. The existing effective method for the quantitative detection of probiotics is a flat plate counting method, and the method has the defects of long time consumption (generally 72 hours), large workload, more complicated operation and the like in practical application. In recent years, molecular biology techniques represented by Real-time quantitative PCR (qPCR) have been widely applied to quantitative detection of probiotics. Compared with the traditional culture method, the qPCR method is rapid, sensitive and specific, but the method belongs to relative quantification, the accuracy and repeatability of the result depend on the quality of a drawn standard curve and the amplification efficiency to a great extent, and a nucleic acid standard with known concentration is required. A new micro-Droplet Digital PCR (ddPCR) is considered as a third generation nucleic acid amplification technology, DNA molecules diluted to a certain concentration are distributed into 10000-20000 micro-droplets, the number of the DNA molecules in most micro-droplets is 1 or 0, then the number of positive reaction units is read through PCR amplification and positive signal accumulation, the proportion of positive signals and negative signals is collected, and the number of the DNA molecules in a sample is calculated according to Poisson distribution, so that the absolute quantification of the DNA molecules is realized.
At present, the technology is increasingly applied to the field of food, and is mainly applied to the aspects of food-borne pathogenic bacteria detection, transgenic plant detection, animal-borne component detection and the like. The invention aims to establish a rapid and accurate quantitative method for common probiotics in infant food by adopting a ddPCR detection technology, and provides thinking and technical support for formulating a quantitative detection standard for the probiotics in the infant food.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a rapid, sensitive and accurate quantitative detection method for bifidobacteria in milk powder.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a microdroplet digital PCR method for detecting bifidobacteria in milk powder comprises a pretreatment step of a sample to be detected, wherein the pretreatment step comprises the following steps:
(1) centrifuging the solution of the to-be-detected product, and removing a supernatant;
(2) carrying out cold and hot alternate treatment;
(3) digesting with proteinase K;
(4) digesting with lysozyme;
(5) digestion was performed with proteinase K and buffer GA.
The acquisition of template DNA with high quality and accurate concentration is a determining factor for accurate quantification of ddPCR, and because bifidobacteria are gram-positive bacteria, the cell walls are not easy to break, and the infant formula milk powder contains a large amount of protein molecules and has complex components, the difficulty in extracting the template DNA with high quality is increased, the invention specifically optimizes the extraction methods of the bifidobacteria DNA and the bifidobacteria in the infant formula milk powder respectively.
By adopting the method, fat and macromolecular substances which interfere with detection in the milk powder can be effectively removed, and effective denaturation and digestion of protein in the milk powder can be realized through cold-hot alternate treatment and protease treatment, so that the quality of subsequent extracts is further ensured.
The buffer solution GA is a commercial product, and comprises Tris-HCL with the concentration of less than or equal to 20mM, EDTA with the concentration of less than or equal to 2mM and SDS with the concentration of less than or equal to 10%, and the pH value is 7.6-8.4.
In the invention, the step (2) is specifically as follows: treating at the temperature of minus 18 ℃ to minus 20 ℃ for 1.5 to 2min, then treating at the temperature of 100 ℃ for 0.9 to 1.1min, and repeating for multiple times.
In the invention, the digestion conditions in the step (5) are 55-57 ℃ and 1.2-1.5 h.
The invention increases the condition of further enzymolysis of mycoprotein in the conventional treatment, and can ensure better subsequent quantitative extraction effect of nucleic acid.
In the invention, the step (1) specifically comprises two times of centrifugation, wherein the first time of centrifugation is as follows: centrifuging at 11,000-12,000 rpm for 10-12 min; the second centrifugation is: and mixing the precipitate obtained by the first centrifugation with PBS, and centrifuging for 10-12 min at 11,000-12,000 rpm.
In the invention, in the step (3), the mass ratio of the protease K to the to-be-detected product is (0.015-0.30): 1;
and/or in the step (5), the mass ratio of the protease K to the buffer GA is 1: (0.1-0.2).
In the invention, the primers and probes used by the microdroplet digital PCR method are shown in SEQ ID No. 1-3.
In the present invention, the reaction system of the microdroplet digital PCR method comprises, per 20. mu.L: 10 mu L of ddPCR premix solution by 2 Xprobe method, 500nM final concentration of upstream and downstream primers, 250nM final concentration of probe, 2 mu L of template, and the balance of sterile double distilled water.
In the invention, the reaction procedure of the microdroplet digital PCR method is as follows: 10min at 95 ℃; 43 cycles of 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 30 s; 10min at 98 ℃.
In the invention, during the development of PCR reaction, the specific single copy gene of the bifidobacterium is firstly determined, and the specific primer probe is developed, so that the accuracy of ddPCR detection results is reduced due to the change of quantitative copy number caused by multi-copy genes. Secondly, the ddPCR detection principle is that the copy number of DNA molecules is calculated by the number of positive micro-drops and the number of negative micro-drops by using a Poisson distribution formula, so the accuracy of ddPCR and the amplification efficiency of PCR can be reflected by the strength of a fluorescence signal, and therefore, the concentration of a primer probe in ddPCR reaction and the annealing temperature of ddPCR reaction conditions are optimized.
Finally, the invention completely establishes a quantitative detection method for the bifidobacteria in the (infant formula) milk powder by applying a Droplet Digital PCR (Droplet Digital PCR, ddPCR) technology. The primer probe is designed by taking the single copy specific gene rpsl of the bifidobacterium as a target gene, the ddPCR reaction condition is optimized, the specificity, the sensitivity and the repeatability of the method are investigated, the detection result is verified by comparison with a plate counting method, and the result shows that the minimum detection limit of the bacterial pure culture solution is 102CFU/g, the minimum detection limit of the simulated sample is 103CFU/g, does not have cross reaction with 10 kinds of lactobacillus, and has better repeatability. The established ddPCR and plate counting method is adopted to detect 10 batches of infant formula milk powder samples sold in the market, the deviation of the quantitative lg value of the two methods is between +/-0.5 lg,the results were consistent. The ddPCR method established by the invention can be used for quantitatively detecting the bifidobacteria in the infant formula milk powder more quickly, sensitively and accurately, and has a certain application prospect.
The invention also provides an application of the microdroplet digital PCR method in the quality control of the milk powder.
Preferably, the milk powder is infant formula milk powder.
The invention has the beneficial effects that:
the invention applies the ddPCR detection method to the detection of bifidobacteria in infant formula milk powder for the first time, and the minimum detection limit of the established ddPCR detection method to the bifidobacteria bacteria pure culture solution is 102CFU/g, the minimum detection limit of the simulated sample is 103CFU/g, and 10 batches of infant formula milk powder samples sold in the market are detected, the deviation of the quantitative lg values of the two methods is within +/-0.5 lg, and the results are good in consistency. The method for quantitatively detecting the bifidobacteria in the existing effective food is a flat plate counting method which is generally about 3 days, can reduce the detection period to about 1 day, has the advantages of good specificity, high accuracy and the like, and is suitable for quantitatively detecting the bifidobacteria in milk powder products (infant formula).
Drawings
FIG. 1 shows the results of the primer probe screening experiment.
FIG. 2 shows the result of the primer probe specificity verification. Wherein, 1-bifidobacterium bifidum CICC6071, 2-bifidobacterium infantis CICC6069, 3-bifidobacterium longum CGMCC1.2186, 4-bifidobacterium adolescentis CICC6070, 5-bifidobacterium animalis Bb-12, 6-bifidobacterium lactis HN019, 7-bifidobacterium breve CGMCC1.2213, 8-lactobacillus CICC21800, 9-lactobacillus delbrueckii CICC6047, 10-lactobacillus paracasei CICC20262, 11-lactobacillus rhamnosus LGG, 12-lactobacillus salivarius CICC23174, 13-lactobacillus johnsonii CICC6084, 14-lactobacillus acidophilus CICC6081, 15-lactobacillus delbrueckii subspecies CICC20708, 16-lactobacillus rapi CICC6032 and 17-lactobacillus thermophilus CICC 21728.
FIG. 3 shows the results of the optimization of primer probe concentration. Wherein, 1-the first group, 2-the second group and 3-the third group.
Fig. 4 shows the annealing temperature optimization results.
FIG. 5 is a gradient dilution pure bacteria solution ddPCR amplification oil drop distribution diagram.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1. Materials and methods
1.1 materials and reagents
TABLE 1 list of bacteria for experiments
MRS culture medium: BD corporation, usa; phosphate Buffered Saline (PBS): american Sammer Federation; cetyl trimethylammonium Bromide (CTAB), Triton X-100, ethylenediaminetetraacetic Acid (EDTA): sigma corporation, usa; sodium hydroxide and sodium chloride were analytically pure: chemical agents of the national drug group, ltd; bacterial genomic DNA extraction kit (centrifugal column type), proteinase K (20mg/ml), lysozyme (50 mg/ml): beijing TIANGEN corporation; the micro-drop digital PCR reaction system comprises the following reagents: burle corporation, usa; the primers and probes were synthesized by Biotechnology engineering (Shanghai) Ltd.
1.2 instruments and devices
Q × 200 droplet digital PCR instrument: burle corporation, usa; a PCR instrument: ABI, USA; high-speed centrifuge, heating blending appearance: eppendorf, Germany; sterile homogenizer: shanghai Bilang instruments, Inc.; constant temperature and humidity incubator: MMM company, Germany; the biological safety cabinet: thermo corporation, usa.
1.3 methods
1.3.1 ddPCR reaction system and detection method
(1) Preparing a ddPCR system, wherein the system is as follows: 2 XProbe method ddPCR premix 10 u L, will be diluted to 10 u M primer and probe, respectively will be 2.25 u L upstream, downstream primer and 1.0 u L probe add system, add 2 u L template, finally with the double distilled water to make up 20u L.
(2) And transferring the fully-mixed ddPCR system into a microdroplet generation card, adding 70 mu L of microdroplet generation special oil into the microdroplet generation card, and putting the microdroplet generation card into a microdroplet generator for reaction. The resulting 40. mu.L of the microdroplet was then transferred to a 96-well plate for ddPCR, and the 96-well plate was sealed with a membrane sealer to prepare for PCR.
(3) Reaction procedure for ddPCR: 10min at 95 ℃; 43 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; 10min at 98 ℃.
(4) After ddPCR is finished, the 96-well plate is placed into a QX200 Droplet Reader instrument, sample information is recorded in sequence, and a final result (copy/20 mu L) is calculated according to Quantasoft software.
1.3.2 primer Probe design, screening and specificity experiments
According to the screened single copy gene rpsL (ID:1022640) of the bifidobacterium, a Primer probe sequence is designed on the gene, a Primer Premier 6.0 is used for designing a Primer probe, verification is carried out through Oligo 7.37, BLAST online comparison is carried out, three pairs of Primer probes are designed, and as shown in Table 2, the 5 'end of each probe is marked with FAM, and the 3' end of each probe is marked with BHQ. And (3) taking the DNA of the bifidobacterium lactis HN019 as a template, and screening three pairs of primer probes by using a ddPCR method.
A pair of primer probes obtained by screening the above-mentioned strains were tested for the homologous and homologous species of Bifidobacterium by ddPCR detection method described in section 1.3.1 using DNA of 7 Bifidobacterium strains and 10 Lactobacillus strains in Table 1 as templates to verify the specificity of the primer probes.
TABLE 2 ddPCR primer/Probe sequences (SEQ ID Nos. 1-9)
1.3.3 ddPCR reaction Condition optimization
1.3.3.1 optimization experiment of primer and probe concentration
Three sets of upstream and downstream primers and probe concentrations to be optimized are set, as shown in table 3, the DNA of bifidobacterium lactis HN019 is used as a template, a pair of primer probes obtained by the screening are used for carrying out ddPCR detection on the three sets of concentrations respectively according to the records in part 1.3.1, and the optimal primer probe ddPCR reaction concentration is screened according to the result generated by a microdroplet detector.
TABLE 3 three different primer Probe concentrations
1.3.3.2 ddPCR annealing temperature optimization experiment
In designing the reaction procedure for ddPCR amplification, the annealing temperature of ddPCR was optimized based on the ddPCR reaction system described in section 1.3.1, using the DNA of Bifidobacterium lactis HN019 as a template and using the pair of primer probes and the corresponding concentrations obtained by the above screening. Setting the annealing temperature to 53 ℃, 53.5 ℃, 54 ℃, 55 ℃, 56 ℃ and 58 ℃ for gradient ddPCR, and screening out the optimal annealing reaction temperature according to the result generated by a microdroplet detector.
1.3.4 nucleic acid extraction method
1.3.4.1 Standard Strain nucleic acid extraction method optimization
Nucleic acid extraction is carried out on stock solution of the Bifidobacterium lactis HN019 culture by using a rhizobacteria genome DNA extraction kit (centrifugal column type), and the extraction method is optimized. Selection of the culture stock solution 10-1～10-3Extracting nucleic acid with diluted bacteria solutionPlate counting is carried out on the bacterial liquid according to a standard method of GB4789.34-2016 (national food safety standard food microbiology inspection) for detecting bifidobacteria, meanwhile, an established ddPCR method is applied to quantitatively detect the extracted nucleic acid, each experiment is repeated for 3 times, the result is the average value of 3 experiments, and the optimal extraction method is selected.
The optimization method 1: 1mL of stock solution of the Bifidobacterium lactis HN019 culture is placed in a centrifuge for 5min at 12,000rpm, the supernatant is poured off, the condition that lysozyme solution is added in the kit method is that the final concentration is 20mg/mL, the lysozyme is digested for 1.5h at 37 ℃, the final concentration of the lysozyme is increased to 50mg/mL by the optimization method, other conditions are unchanged, and then nucleic acid extraction is carried out according to the kit instruction.
The optimization method 2 comprises the following steps: on the basis of optimizing the conditions of the method 1, the step of adding 20 mu L of proteinase K for digestion after the step of lysozyme treatment in the kit method is further changed into the step of adding 50 mu L of proteinase K and adding 150 mu L of buffer GA provided by the kit, the digestion is carried out for 1.5h at 56 ℃, other conditions are unchanged, and then the nucleic acid extraction is carried out according to the kit instructions.
1.3.4.2 optimization of nucleic acid extraction method for simulated milk powder sample
Because the milk powder sample contains a large amount of macromolecular proteins and fats, the traditional nucleic acid extraction method is difficult to adopt, so the extraction method is optimized. Weighing two segments of 5.0g of commercially available infant formula powder of certain domestic brand without adding Bifidobacterium, placing in a homogenizing bag containing 44.0mL of normal saline, anaerobically culturing Bifidobacterium HN019 suspension 10 at 37 deg.C for 48 hr-1～10-31ml of each diluted bacterial solution is beaten by a beating type homogenizer for 2min to prepare a sample uniform solution with the volume ratio of 1: 10. And (3) extracting nucleic acid from the uniform solution of each dilution simulation sample, simultaneously carrying out plate counting on the bacterial solution according to the method recorded in 1.3.4.1, quantitatively detecting the extracted nucleic acid by applying the optimized and established ddPCR method, and repeating each experiment for 3 times to select the optimal extraction method.
Method 1 (enzymatic hydrolysis plus CTAB method): adding 100 mu L proteinase K into 1mL of the simulated sample, and digesting for 3h at 56 ℃; centrifuging at 12,000rpm for 5min, collecting the intermediate clear liquid, and extracting DNA by CTAB method.
CTAB DNA extraction method: adding 800 μ of LCTAB solution (40mg CTAB, 23.4mg NaCl) into the sample to be extracted, and heating at 65 deg.C for 4 h; adding 8 μ LRNase, standing at room temperature for 5 min; violently shaking the centrifugal tube for 1min, and then centrifuging at 12,000rpm for 10 min; transferring the supernatant into another centrifuge tube, adding an equal volume of chloroform solution, shaking up, and centrifuging at 12,000rpm for 5 min; transferring the supernatant into another centrifuge tube, adding 3/4 isopropanol solution of the volume of the supernatant, and standing at room temperature for 5 min; centrifuging at 12,000rpm for 10min, discarding supernatant, adding 500 μ L75% ethanol solution, centrifuging at 12,000rpm for 2min, and repeating once; removing supernatant, standing at room temperature for 10min, and air drying; about 100. mu.L of TE solution was added to the centrifuge tube to dissolve the DNA.
Method 2 (alkaline plus CTAB method): 1mL of the simulated sample was added with 200. mu.L of a solution (4mg NaOH, 3.8mg EDTA), and heated at 100 ℃ for 10 min; the DNA was extracted from the intermediate supernatant by the CTAB method described above.
Method 3 (enzyme method plus optimized kit extraction method): 1mL of the mock sample was centrifuged at 12,000rpm for 10 min; removing supernatant and fat, adding 1ml PBS and centrifuging at 12,000rpm for 10 min; standing the precipitate obtained by secondary centrifugation at-20 deg.C for 1.5min in a refrigerator; standing at 100 deg.C for 1min, and treating the sample repeatedly for 6 times; adding 200 μ L LPBS solution, digesting with 100 μ L proteinase K +1 μ L LTriton X-10056 deg.C for 1.5 h; centrifuging at 12,000rpm for 5min to remove supernatant, adding 200 μ L lysozyme, and digesting at 37 deg.C for 1.5 hr; adding 50 μ L proteinase K and buffer GA150 μ L provided by the kit, digesting at 56 deg.C for 1.5h, and extracting nucleic acid according to the kit instructions.
1.3.5 plate counting and ddPCR detection of Bifidobacterium pure culture
1.3.5.1 plate count
Selecting proper dilution from stock solution of the culture of the bifidobacterium lactis HN019 to count plates, repeating each dilution for 2 times according to a standard method of GB4789.34-2016 (national food safety standard food microbiology inspection for bifidobacterium inspection), placing the plates at 37 ℃ for anaerobic culture for 48-72 hours, and observing the growth condition of colonies. And selecting the plates with the colony number of 30-300 CFU and no spread colony growth to count the total number of the colonies, wherein the colony number of each dilution degree is an average value of the counting results of the two plates.
1.3.5.2 detection of Bifidobacterium culture by ddPCR
Culturing Bifidobacterium lactis HN019 stock solution at 37 deg.C for 48 hr under anaerobic condition to 10%-6Diluting the bacterial liquid, extracting DNA by applying a bifidobacterium nucleic acid extraction method established by 1.3.4.1, detecting by applying an established ddPCR detection method, repeating the test for 3 times for each concentration gradient detection, performing statistical analysis by means of SPSS14.0, and calculating the CV value. And (3) according to the concentration of the bacterial liquid quantified by the copy number, taking lg fixed value, comparing the lg fixed value with a plate count lg fixed value, determining a quantitative range, and obtaining the lowest detection limit.
The Copy number of the target gene (Copy/20. mu.L) per reaction can be directly measured by ddPCR, and the conversion formula of the Copy number of ddPCR and the concentration of the corresponding bacterial suspension is as follows:
c-concentration of bacterial suspension (CFU/mL) is obtained according to conversion of Copy number of ddPCR; copy number of 20ul system in X-ddPCR (pieces/20. mu.L); v1-final volume of constant volume (μ L) for DNA extraction; v2-volume of suspension of DNA extracted (mL); volume of DNA template (. mu.L) in V3-ddPCR reaction.
1.3.5.3 plate count and ddPCR detection of probiotic simulated infant formula milk powder samples
Weighing two segments of 5.0g of commercially available infant formula powder of certain domestic brand without adding Bifidobacterium, placing in a homogenizing bag containing 44.0mL of normal saline, anaerobically culturing Bifidobacterium HN019 suspension 10 at 37 deg.C for 48 hr-1～10-51mL of each diluted bacterial solution is beaten for 2min by a beating homogenizer to prepare a sample uniform solution with the volume ratio of 1: 10, 3 samples are repeated at each adding level, 1 part of sterile weighed sample 5g of infant milk powder sample is added with 45mL of normal saline to replace the added bacterial solution as a negative reference, and the 5 added levels of infant formula milk powder sample solutions are respectively diluted in a gradient manner to carry out plate counting. Simultaneously, 1mL of each sample solution is taken, DNA is extracted by the method for extracting the nucleic acid of the simulated milk powder established by 1.3.4.2,the nucleic acids were finally dissolved in 100. mu.LTE buffer and ddPCR detection was performed.
The concentration of the bacterial liquid corresponding to the detection result of ddPCR on the sample can be obtained by converting the formula (1) in 1.3.5.2, and the lg fixed value of the quantitative result is compared with the lg fixed value of the plate count.
1.3.6 plate count and ddPCR detection of actual infant formula milk powder samples
The method comprises the steps of extracting genome DNA from 10 commercially available infant formula milk powder of different domestic manufacturers by a two-stage method of 1.3.4.2, carrying out quantitative detection according to an optimized ddPCR method, taking 2 parallel samples from each sample, repeating the samples for 3 times, simultaneously carrying out quantitative detection on the samples by a plate counting method (GB4789.34-2016 (Standard method for food safety national Standard food microbiology inspection) on bifidobacterium detection), and comparing the consistency of quantitative results of the two methods.
2 results and analysis
2.1 primer Probe screening and specificity verification
The three pairs of primer probes in table 2 were screened by ddPCR, and it can be seen from FIG. 1 that the first set of primer probes showed positive oil drop signals, and the other two sets showed negative non-amplified results, so the first set of primer probes was selected for the subsequent experiments. The result of ddPCR amplification using the first set of primer probes selected from the DNA of Bifidobacterium strains and Lactobacillus strains of 7 and 10 in Table 1 as templates by the method described in section 1.3.1 is shown in FIG. 2, where all Bifidobacterium strains of 7 show positive oil drop signals, and all other Lactobacillus strains of 10 show no negative amplification, indicating that the first set of primer probes selected from Bifidobacterium strains has good specificity.
2.2 primer Probe concentration Excellent results
The result of ddPCR amplification of 3 primer probe sets with the DNA of Bifidobacterium lactis HN019 as a template and with reference to the description in section 1.3.1 is shown in FIG. 3, the fluorescence signal intensity of ddPCR amplification of three primer probe sets with concentrations is not obviously different, and the fluorescence amplitude difference between negative and positive droplets is not obvious, so that the primer probe concentration most suitable for practical application is selected as the third primer probe set: 500nM, 250 nM.
2.3 annealing temperature optimization results
The annealing temperature of the ddPCR amplification system is selected to be 53 ℃, 53.5 ℃, 54 ℃, 55 ℃, 56 ℃ and 58 ℃ for optimization experiment, the result is shown in figure 4, the annealing temperature has fluorescence signals detected within the range of 53-58 ℃, the negative and positive droplets have obvious boundaries, the amplification amplitude between the negative and positive oil drops is gradually increased, the maximum amplification amplitude is reached at 58 ℃, and the number of dispersed oil drops between the negative and positive oil drops under the temperature condition indicates that the non-specific amplification of ddPCR is less, so 58 ℃ is selected as the optimal annealing temperature for reaction.
2.4 optimization of Bifidobacterium nucleic acid extraction method
Carrying out gradient dilution on the Bifidobacterium lactis HN019 pure culture solution and applying a plate counting method to obtain the original bacterial solution with the concentration of 1.2 multiplied by 107And CFU/mL, and detecting the result of the nucleic acid extraction optimization method by applying the ddPCR method obtained by optimization. The results are shown in table 4, after the concentration of lysozyme is increased by more than 1 time by the optimization method 1, the extraction effect is improved to a certain extent, but the difference between the detection result and the plate counting result is 1 order of magnitude; the optimization method 2 changes the digestion condition of the proteinase K on the basis of increasing the concentration of the lysozyme, greatly improves the nucleic acid extraction effect, and the detection result of the ddPCR method shows that the method has good extraction effect on 3 dilutions of the strain stock solution and is consistent with the quantitative result of the plate counting method, so that the nucleic acid extraction method is used for the subsequent ddPCR detection experiment.
TABLE 4 optimization of Bifidobacterium nucleic acid extraction Process
2.5 simulated milk powder sample nucleic acid extraction method optimization
To 10-1～10-3The plate count of the diluted bacteria liquid simulation sample is 1.4 multiplied by 10 respectively7、1.5×106、1.3×105CFU/mL. And simultaneously, detecting the three nucleic acid extraction methods by applying the optimized ddPCR method. The results are shown in Table 5, from which it is clear that the extraction effect of method 1 is not satisfactoryNucleic acid detection, the extraction effect of the method 2 is improved to a certain extent compared with the extraction effect of the method 1, but the quantitative result is different from the plate counting result by 1 order of magnitude, the method 3 adopts an enzyme method to pretreat the simulation sample and combines the optimized kit extraction method, the result shows that the extraction effect is greatly improved, the ddPCR detection quantitative result is consistent with the plate counting result, and the method 3 is used as the extraction method of the nucleic acid sample in the milk powder sample for subsequent experiments.
TABLE 5 optimization of simulated milk powder sample nucleic acid extraction method
2.6 ddPCR detection and plate counting of Bifidobacterium pure culture
Carrying out gradient dilution on the Bifidobacterium lactis HN019 pure culture solution and applying a plate counting method to obtain the original bacterial solution with the concentration of 1.1 multiplied by 108CFU/mL, quantitative lg value of 8.05. ddPCR was performed to dilute the original bacteria solution to 10 degrees-6Detection, ddPCR at 10-1The concentration of the dilution template is too high, no negative oil drop exists, the concentration of the template cannot be calculated by an instrument, and the dilution template can be determined only by proper dilution. The results are shown in FIG. 5, 10-2To 10-6Positive oil drops can be detected by the ddPCR of the diluted bacterium liquid, the quantitative result is shown in table 6, the fixed value result of the original bacterium suspension (bacterium stock solution) obtained by ddPCR detection is 8.13-8.47, the CV values are 4.31-12.76 and are less than 20%, the repeatability is good, the difference range of the fixed value result and the plate counting quantitative lg value is 0.08-0.42, the deviation is +/-0.5 lg, and the consistency of the fixed value result and the plate counting quantitative result is good. At 10-6The diluted bacterial liquid reaches the lowest detection limit of the ddPCR method, and the quantitative result of the bacterial liquid is 296 CFU/g.
TABLE 6 results of ddPCR quantification of pure inoculum solution
2.7 plate count and ddPCR detection of probiotic simulated infant formula milk powder samples
10-1～10-5The plate count results of the infant formula milk powder samples at the dilution bacterial suspension addition level were 3.5X 10 respectively7、3.6×106、4.3×105、3.3×104、3.8×103CFU/g. The results of the ddPCR method on the quantification of bifidobacteria added to infant formula milk powder food matrices are shown in Table 7 at 103～107The CV value in the concentration range of the CFU/g sample is 3.92-15.80%, the repeatability is good, the difference range of the quantitative lg value and the plate counting quantitative lg value is 0.03-0.28, the deviation is +/-0.5 lg, and the consistency with the plate counting quantitative result is good.
TABLE 7 Probiotics sample simulated ddPCR and plate count quantification results
2.8 plate counting and ddPCR detection of actual infant formula milk powder samples
Carrying out plate counting quantitative detection on bifidobacterium on 10 commercially available infant formula milk powder second segments (A1-A8) of different domestic manufacturers, wherein the detection results all achieve that the number of viable bacteria is not less than 106CFU/g, substantially in accordance with the label. Meanwhile, the established ddPCR method is used for detection, the results are shown in Table 8, and the quantitative detection results of the samples are all more than 106CFU/g, and the deviation of the quantified lg value and the plate counting lg value is within +/-0.5 lg, and the result consistency is better. Therefore, the ddPCR quantitative detection method for the bifidobacteria in the infant formula milk powder, which is established by the invention, can be applied to the quantitative detection of the bifidobacteria in actual samples.
TABLE 8 detection results of ddPCR and plate count for commercially available samples
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (10)
1. A microdroplet digital PCR method for detecting bifidobacteria in milk powder is characterized by comprising a pretreatment step of a sample to be detected, wherein the pretreatment step comprises the following steps:
(1) centrifuging the solution of the to-be-detected product, and removing a supernatant;
(2) carrying out cold and hot alternate treatment;
(3) digestion with proteinase K;
(4) digesting with lysozyme;
(5) digestion was performed with proteinase K and buffer GA.
2. The microdroplet digital PCR method according to claim 1, wherein step (2) is specifically: treating at the temperature of minus 18 ℃ to minus 20 ℃ for 1.5 to 2min, then treating at the temperature of 100 ℃ for 0.9 to 1.1min, and repeating for multiple times.
3. The microdroplet digital PCR method of claim 1 or 2, wherein the digestion in step (5) is performed at 55-57 ℃ for 1.2-1.5 h.
4. Microdroplet digital PCR method according to any of claims 1-3, characterized in that step (1) comprises in particular two centrifugations, the first centrifugation being: centrifuging at 11,000-12,000 rpm for 10-12 min; the second centrifugation is: and mixing the precipitate obtained by the first centrifugation with PBS, and centrifuging for 10-12 min at 11,000-12,000 rpm.
5. The microdroplet digital PCR method according to any one of claims 1-4, wherein in step (3), the mass ratio of the proteinase K to the sample is (0.015-0.30): 1;
and/or in the step (5), the mass ratio of the protease K to the buffer GA is 1: (0.1-0.2).
6. The microdroplet digital PCR method of any one of claims 1-5, wherein primers and probes used in the microdroplet digital PCR method are shown in SEQ ID Nos. 1-3.
7. The microdroplet digital PCR method according to any one of claims 1 to 6, wherein the reaction system of the microdroplet digital PCR method comprises, per 20 μ L: 10 mu L of ddPCR premix solution by 2 Xprobe method, 500nM final concentration of upstream and downstream primers, 250nM final concentration of probe, 2 mu L of template, and the balance of sterile double distilled water.
8. The microdroplet digital PCR method of any one of claims 1-7, wherein the reaction sequence of the microdroplet digital PCR method is: 10min at 95 ℃; 43 cycles of 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 30 s; 10min at 98 ℃.
9. Use of a microdroplet digital PCR method according to any one of claims 1 to 8 in the quality control of milk powder.
10. Use according to claim 9, wherein the milk powder is an infant formula.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140199697A1 (en) * | 2013-01-16 | 2014-07-17 | Texas Tech University System | Quantification and molecular detection of lactic acid bacteria in a sample |
| CN105695560A (en) * | 2014-11-25 | 2016-06-22 | 上海市质量监督检验技术研究院 | Method for identifying Bifidobacterium lactis in infant formula milk powder |
| CN112626246A (en) * | 2021-01-04 | 2021-04-09 | 扬州大学 | Detection method for identifying three bacteria in milk through multiple PCR |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140199697A1 (en) * | 2013-01-16 | 2014-07-17 | Texas Tech University System | Quantification and molecular detection of lactic acid bacteria in a sample |
| CN105695560A (en) * | 2014-11-25 | 2016-06-22 | 上海市质量监督检验技术研究院 | Method for identifying Bifidobacterium lactis in infant formula milk powder |
| CN112626246A (en) * | 2021-01-04 | 2021-04-09 | 扬州大学 | Detection method for identifying three bacteria in milk through multiple PCR |
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| Title |
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| 卢海强等: "数字PCR在食源性致病菌检测中的应用 进展", 生物技术进展, vol. 11, no. 3, pages 264 * |
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