CN113999925A - Primer combination and application thereof in detection of lactobacillus paracasei in dairy products - Google Patents
Primer combination and application thereof in detection of lactobacillus paracasei in dairy products Download PDFInfo
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Abstract
The invention discloses a primer combination and application thereof in detecting lactobacillus paracasei in dairy products, wherein the primer combination is based ongyrB gene, by establishing a phylogenetic tree of lactobacillus paracasei, analyzing the characteristics of lactobacillus paracasei strains, and designing a specific primer combination; the designed primer combination is applied to a real-time fluorescent quantitative loop-mediated isothermal amplification (qLAMP) technology, so that the detection of the lactobacillus paracasei in the dairy product is realized. The primer combination has strong specificity and high sensitivity, and can quickly detect lactobacillus paracasei in dairy products. The method is suitable for qualitative and quantitative detection of lactobacillus paracasei in dairy products.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and relates to a primer and a method for detecting bacteria, in particular to a primer and a corresponding detection method for detecting lactobacillus paracasei in dairy products.
Background
Lactobacillus paracasei (l.paracasei), belonging to the phylum firmicutes, class baculomycetes, order lactobacillales, family lactobacillaceae, species lactobacilli, belonging to gram-positive bacteria, is commonly used as a starter or an auxiliary starter for milk and dairy products, is a probiotic bacterium, a lactic acid bacterium, which has been intensively studied in recent years, and after being digested by the gastrointestinal tract of a human body, lactobacillus paracasei can still survive in the intestinal tract and exert the probiotic effects of promoting the digestive absorption of the human body, regulating the immunity and improving the intestinal microecological balance.
The plate counting method is a traditional colony counting method, is subjectively influenced, tedious and complicated, time-consuming and labor-consuming, has large errors, can not distinguish viable but non-culturable bacterial cells in a net month, can not effectively quantify a single strain in a complex strain, and particularly when detection occurs in a fermentation system with a complex microbial community, is difficult to quantify microorganisms by the plate counting method, particularly at the species level.
Quantitative loop-mediated isothermal amplification (qLAMP) is a microbial quantitative analysis method with high sensitivity and strong specificity, fluorescent dyes such as SYBR Green I and the like are added into an LAMP reaction system, the increase of the fluorescent intensity is in direct proportion to the quantity of double-stranded DNA generated by the LAMP system in the amplification reaction process, and the quantitative analysis of the double-stranded DNA is realized according to the correlation between the fluorescent dye intensity and the quantity of the double-stranded DNA. Because of the stability of DNA molecules, DNA molecules do not decompose automatically after microbial bacteria die and lyse, it is difficult to determine whether positive amplification results are viable or dead when various nucleic acid amplification techniques are used. Therefore, a detection method capable of distinguishing a non-viable bacterium from a viable bacterium is urgently required.
The monoaziridine (PMA) is a DNA molecular nucleic acid dye, can only enter dead cells with incomplete cell membranes, and inhibits amplification reaction, so that the specificity quantification of live cells can be realized by combining the PMA with qLAMP. However, the research of rapidly and accurately detecting lactobacillus paracasei in fermented milk by combining PMA and qLAMP has not been reported.
Disclosure of Invention
The invention aims to provide a primer combination, which is designed based on the gyrB gene and the strain characteristics of lactobacillus paracasei and achieves the purposes of being applied to a quantitative loop-mediated isothermal amplification (qLAMP) technology, and specifically, sensitively and rapidly detecting lactobacillus paracasei in dairy products;
the invention also aims to provide the application of the primer combination for detecting lactobacillus paracasei in dairy products.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a primer combination is formed by combining an outer primer, an inner primer and a loop primer, and is a primer combination based on a gyrB gene, wherein the outer primer, the inner primer, the loop primer and sequences thereof are respectively as follows:
outer primer FOP: 5'-GGCTATGTCTACAT-3',
An outer primer BOP: 5'-GCGATACCCGCA-3',
Inner primer FIP: 5'-GCAACTGGCCTAGAATGTTACGCCAAGGCAAAATGACG-3',
The inner primer BIP: 5'-CGTTACAAAGGGCTTCCGGATCCATGGTCGTCT-3',
Loop primer FLP: 5'-TTCTTCATCGGAATCGA-3' and
loop primer BLP: 5'-GAAATGGACGCCAAC-3' are provided.
The invention also provides application of the primer combination in detecting lactobacillus paracasei in dairy products.
The method is limited by using the primer combination as a primer of a qLAMP reaction system, using DNA of the dairy product to be detected after PMA treatment as a template of the qLAMP reaction system to carry out qLAMP reaction, monitoring the fluorescence intensity of the qLAMP reaction system in real time, if the reaction result is positive, the dairy product to be detected contains lactobacillus paracasei, and if the reaction result is negative, the dairy product to be detected does not contain lactobacillus paracasei.
As a further limitation, the DNA of the dairy product to be detected is extracted by the following steps:
s11, adding water into the dairy product to be detected for dissolving/diluting, culturing in an MRS culture medium, and then washing cells with sterile normal saline to obtain a liquid alpha to be detected;
s12, adding a PMA solution into the bacterial suspension alpha to obtain a reaction liquid beta, carrying out photophobic reaction on the reaction liquid beta, incubating under a halogen light source, centrifuging, discarding the supernatant, and taking bacterial cells to wash and suspend to obtain a bacterial suspension gamma;
and S13, carrying out boiling water bath treatment on the bacterial suspension gamma, cooling, centrifuging, and taking supernatant to obtain the DNA of the dairy product to be detected.
As a further limitation, in step S11, the culturing is carried out at 35-40 deg.C for 20-30 h.
In a further more limited aspect, in step S12, the PMA solution is at a concentration of 10mg/mL;
the concentration of lactobacillus paracasei in the reaction liquid beta is 5 mug/mL;
the reaction is carried out in a dark place for 3-8 min;
the power of the halogen light source is 400-600 w;
the incubation time is 8-15 min;
the centrifugation is carried out at 10000-15000rpm for 1-3 min.
As a further limitation, in step S13, the boiling water bath treatment is carried out for 8-15 min;
the temperature after cooling is 20-30 ℃;
the centrifugation is carried out at 10000-15000rpm for 8-15 min.
As another limitation, the qLAMP reaction system comprises, by volume, 3-4 parts of inner primer FIP, 3-4 parts of inner primer BIP, 0.3-0.8 part of outer primer FOP, 0.3-0.8 part of outer primer BOP, 3-5 parts of loop primer FLP, 3-5 parts of loop primer BLP, 0.8-1.2 parts of template, 2-3 parts of 10 multiplied reaction buffer, 0.8-1.2 parts of magnesium sulfate solution, 1.1-1.8 parts of dNTPs, 0.2-0.5 part of betaine, 0.8-1.2 parts of polymerase, 0.2-0.5 part of 10000 multiplied SYBRgreenI and 1-1.5 parts of sterile distilled water.
As a further limitation, the reaction buffer is a Thermopol reaction buffer;
the concentration of the magnesium sulfate solution is 40-60 mmol/L;
the concentration of the dNTPs is 8-15 mmol/L;
the concentration of the primer FIP is 8-15 mmol/L;
the concentration of the primer BIP is 8-15 mmol/L;
the concentration of the primer FOP is 8-15 mmol/L;
the concentration of the primer BOP is 8-15 mmol/L.
As a third limitation, the qLAMP reaction system, covered with mineral oil; the qLAMP reaction temperature is 60-65 ℃ and the time is 35-45 min.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
firstly, the primer provided by the invention is obtained by clustering analysis of a gyrB gene sequence on a phylogenetic tree, so that the specificity of lactobacillus paracasei in the dairy product to be detected on the primer is ensured;
secondly, the method for detecting lactobacillus paracasei in the dairy product adopts a method of combining lactobacillus paracasei gyrB gene single copy primer, PMA treatment and qLAMP, can quickly and accurately detect the number of viable bacteria in lactobacillus paracasei in fermented milk, and accurately quantifies the lactobacillus paracasei in actual fermented milk samples containing various microbial strains;
and thirdly, the detection time of the method for detecting the lactobacillus paracasei in the dairy product can be shortened to 120min, and compared with 48h of a flat plate counting method, the detection time is greatly shortened.
The method is suitable for qualitative and quantitative detection of lactobacillus paracasei in dairy products.
The invention is described in further detail below with reference to the figures and the embodiments.
Drawings
FIG. 1 is a phylogenetic tree diagram of Lactobacillus paracasei based on the gyrB gene in example 1;
FIG. 2 shows the results of the primer specificity verification in example 4;
FIG. 3 shows the result of the primer specificity verification in example 10, in which FIG. 3a shows a PMA-qLAMP amplification curve and FIG. 3b shows a PMA-qLAMP dissociation curve;
fig. 4 is an enlarged graph of l.paracasei CGMCC4691 in example 11;
fig. 5 is a detection limit diagram of l.paracasei CGMCC4691 in example 11.
Detailed Description
Example 1 primer for detecting lactobacillus paracasei in dairy products
Principle of primer design
Performing homologous clustering analysis on the amplified sequence by using a phylogenetic tree, and constructing the phylogenetic tree by taking a gyrB gene amplification region sequence of lactobacillus paracasei CGMCC4691 as a target; comparing gyrB sequences of lactobacillus and related species thereof in GenBank by using Clustalx1.81, and selecting 30 sequences;
after 30 sequences are aligned by using DAMBE 4.2.13 software, the same sequences are removed, and a phylogenetic tree is constructed by using the reserved 18 sequences;
based on the gyrB gene, a phylogenetic tree of L.paracasei species is constructed by utilizing a Neighbour-Joining method, a phylogenetic tree diagram is shown in figure 1, and the tree is divided into four main branches, namely a branch I, a branch II, a branch III and a branch IV; branch I contains only one species, l.paracasei (formerly Lactobacillus paracasei); branch II consists of two species, Lactobacillus rhamnosus (l.rhamnosus) and Lactobacillus casei (Lactobacillus casei); branch III consists of two species, namely lacctibacter paraplanarum and latilactibacillus sakei (l.sakei); the branch IV consists of four species, Lactplantibacillus paraplanarum, L.sakei, Lactcaseibacillus zeae, L.rhamnosus.
In a phylogenetic tree, the credibility of L.paracasei gathering in the same branch is 91%, and the gyrB gene can be used for distinguishing lactobacillus paracasei from other species; therefore, a set of species-specific primers for lactobacillus paracasei is designed by aiming at the specific region of the gyrB gene;
(II) primer design
Specific primers were designed using the online primer design software primereplorer V5(https:// primereplorer.jp/elamp 5.0.0/index. html.) based on the intraspecific conserved region and the interspecies variation region of the gyrB gene in Lactobacillus paracasei CGMCC 4691. The sequences of the front outer primer FOP, the rear outer primer BOP, the front inner primer FIP, the rear inner primer BIP, the front loop primer FLP and the rear loop primer BLP are as follows.
Outer primer FOP: 5'-GGCTATGTCTACAT-3',
An outer primer BOP: 5'-GCGATACCCGCA-3',
Inner primer FIP: 5'-GCAACTGGCCTAGAATGTTACGCCAAGGCAAAATGACG-3',
The inner primer BIP: 5'-CGTTACAAAGGGCTTCCGGATCCATGGTCGTCT-3',
Loop primer FLP: 5'-TTCTTCATCGGAATCGA-3' and
loop primer BLP: 5'-GAAATGGACGCCAAC-3'
EXAMPLE 2 determination of the time to Heat-inactivation of Lactobacillus paracasei
The embodiment is an experiment for determining the heat inactivation time of lactobacillus paracasei, and the specific experimental steps are as follows:
suspending 1mL of lactobacillus paracasei in 8 centrifugal tubes to obtain centrifugal tubes X1-X8, treating the centrifugal tubes X1-X8 in water bath at 90 ℃ for 0min,1min,2min,4min,6min,7min,8min or 9min, immediately cooling, and measuring the number of viable lactobacillus paracasei in each centrifugal tube by using a plate counting method;
the results show that the viable cell counts in centrifuge tubes X1-X8 are respectively: 4.5X 108CFU/mL,2.3× 106CFU/mL,1.2×106CFU/mL,4.8×103CFU/mL,1.7×102CFU/mL,6CFU/mL,0CFU/mL, 0CFU/mL, from this, it is known that heat treatment for 8min completely extinguishes Lactobacillus paracasei, so 90 ℃ water bath treatment for 8min was selected for inactivation of the cells.
Example 3 optimization experiment of PMA treatment conditions
This example is an optimization experiment of PMA treatment conditions, in order to obtain the optimal PMA concentration, photophobic reaction time and PMA exposure time to eliminate the influence of dead strain DNA amplification, a three-factor three-level orthogonal experiment was designed according to table 1, each group of lactobacillus paracasei mycelia treated according to table 1 were washed twice with sterile water, resuspended in 100L of sterile water, genomic DNA was extracted by a boiling water bath method, extracted genomic DNA was detected by qLAMP, and the optimal conditions for PMA treatment were determined according to the inhibition rate of PMA on lactobacillus paracasei DNA:
TABLE 1
The specific experimental steps are as follows:
culturing Lactobacillus paracasei CGMCC4691 in MRS culture medium at 37 deg.C for 24 hr, washing cells with sterile physiological saline, and diluting to 10%5CFU/mL, the bacterial suspension was designated V5, and the bacterial suspension was diluted to 108CFU/mL, incubating in 90 ℃ water bath for 8min, determining no viable bacteria by adopting a plate counting method, naming the dead bacteria suspension as D8, collecting proper viable bacteria suspension and dead bacteria suspension, adding a dairy product to be detected, naming a sample as V5+ D8, and correspondingly adjusting the turbidity;
the calculation formula of the bacteriostatic rate is as follows:
Inhibition rate(％)＝[(CV5+D8-Cp)/(CV5+D8-CV5)]×100％
in the formula, CV5+D8:Concentration of L.paracasei without PMA treatment,CFU mL-1； CV5:Concentration of L.paracasei in V5,CFU mL-1；Cp:Concentration of L. paracasei after PMA treatment,CFU mL-1.
K1, K2, K3 are the average values of the inhibition ratios under the respective factors and levels, the average value of the inhibition ratios is generally used to reflect the influence of different levels of the same factor on the test result (inhibition ratio) to determine the optimum level of the factor, and the factors having the same range of the average inhibition ratio of R at each level (range: the average inhibition ratio of the maximum value to the minimum value of the average inhibition ratio) are used to reflect the influence of variation on the test result (inhibition ratio) at the level of each factor; the first and second factors obtained from table 2 are factor a, factor B, and factor C, respectively, wherein the factor that leads to the optimal PMA treatment condition is A2B1C2, and the final result is shown in table 2;
TABLE 2L 9 (3)4) Results of orthogonal experiments
As is clear from Table 2, the optimum PMA treatment conditions were such that the final concentration of PMA was 5. mu.g/mL, the reaction time in the dark was 5min, and the exposure time was 10min, and the inhibition ratio under these conditions was 100%.
Example 4 method for detecting Lactobacillus paracasei in dairy products
The embodiment is a method for detecting lactobacillus paracasei in dairy products, which comprises the following steps of:
firstly) preparation of dairy product to be tested
1) Preparation of 10% skimmed milk powder solution
Adding 10g of skimmed milk powder into 90g of water in a triangular bottle, stirring to dissolve uniformly, sterilizing with high pressure steam at 115 deg.C for 15min, and packaging the 10% skimmed milk powder solution into 9mL sterile test tubes to obtain dairy product to be detected;
2) culturing of the bacteria to be tested
Culturing Lactobacillus paracasei (purchased from China general microbiological culture Collection center with the preservation number of CGMCC4691) in the 10% skimmed milk powder solution at 37 ℃ for 24h to obtain a bacterial suspension M;
3) PMA treatment
Adding 0.5 mu L of PMA solution with the concentration of 10mg/mL into 1mL of bacterial suspension M, adjusting the volume to ensure that the final concentration of the lactobacillus paracasei suspension is 5 mu g/mL respectively, after carrying out dark light-resistant reaction for 5 minutes, respectively exposing test tubes containing the bacterial suspension to 500W of halogen light source, incubating for 10 minutes, centrifuging the exposed bacterial suspension at 12000rpm for 2 minutes at room temperature, discarding the supernatant, washing thallus cells twice with sterile water, and suspending to obtain bacterial suspension N, namely the sample to be detected;
II) detecting lactobacillus paracasei in dairy products
The method comprises the following steps:
s1, extracting DNA of a sample to be detected as a template:
treating the bacterial suspension N treated by PMA in a boiling water bath for 10min by adopting a boiling water bath method, inducing live bacteria to release DNA, cooling to 25 ℃, centrifuging at 12000rpm for 10min, transferring the supernatant to a sterile centrifuge tube, and storing at-20 ℃ for later use;
s2, mixing qLAMP reaction systems, and preparing a system alpha:
based on the primer combination in the embodiment 1, the DNA in the S1 is adopted as a template, and a qLAMP reaction system is prepared by mixing, specifically, the following components are uniformly mixed:
qLAMP reaction system for amplifying gyrB gene in 25 μ L volume, comprising: 2.5. mu.L of 10 × Thermopol reaction buffer (New England Biolabs Inc., USA), 1.0. mu.L of magnesium sulfate solution (50mmol/L), 1.5. mu.L of dNTPs (10mmol/L) (Sigm primer A-Aldrich, St. Louis, MO, USA), 0.3. mu.L of betaine, 3.5. mu.L of primer FIP and primer BIP (10. mu. mol/L), 0.5. mu.L of primer FOP and primer BOP (10. mu. mol/L), 4.0. mu.L of primer FLP and primer BLP (10. mu. mol/L), 1.0. mu.L of 8U of Bst polymerase (New England Biolabs Inc, USA), 0.3. mu.L of 1/700 dilution 10000 × BRSYGreenI, 1. mu.L of LDNA template and 1.4. mu.L of sterile distilled water;
the reaction system is covered and insulated with 20 mul of mineral oil to prevent the evaporation of the amplification reaction solution/qLAMP reagent;
s3, carrying out qLAMP reaction, and monitoring the fluorescence intensity in real time to obtain a reaction result
Covering and insulating the qLAMP reaction system by 20 mu L of mineral oil to prevent the evaporation of the amplification reaction liquid/qLAMP reagent; placing the reaction system at 63 ℃ for reaction by using Applied Biosystems QuantStaudio 3, and monitoring the fluorescence amplification curve in real time for 40 min;
the fluorescence amplification curve was monitored in real time using an Applied Biosystems QuantStudio 3 run at 63 ℃ for 40 minutes;
s4, judging a result: an amplification curve appears in the qLAMP reaction within 40min, and the result is a positive result;
the results of this experiment are shown in FIG. 2;
as can be seen from FIG. 2, the method of this example can be used to detect Lactobacillus paracasei in dairy products.
Example 5-9 detection method of Lactobacillus paracasei in Dairy products
Examples 5 to 9 are methods for detecting lactobacillus paracasei in dairy products, respectively, the steps of which are substantially the same as those of example 4, except for differences in raw material amounts and process parameters, as detailed in table 3:
TABLE 3 summary of the process parameters of examples 5-9
Example 10 verification of the specificity and detection Effect of the primer composition
According to the method of example 4, DNA of 19 strains of common zymophyte, probiotic bacteria and pathogenic bacteria in fermented milk, namely 3 strains of lactobacillus paracasei and 16 strains of non-lactobacillus paracasei (the specific strains are shown in Table 3) are detected by using PMA-qLAMP, all experiments are repeated five times by taking distilled water as a blank control to verify the specificity of the primers;
TABLE 3 information table of strains to be tested
The results are shown in FIG. 3, where FIG. 3a is a PMA-qLAMP amplification curve and FIG. 3b is a PMA-qLAMP dissociation curve;
as can be seen from fig. 3: in a PMA-qLAMP reaction system, compared with 16 strains of non-lactobacillus paracasei, the DNA of the lactobacillus paracasei strain is continuously amplified, the fluorescence intensity (delta Rn) is increased, the blank control has no target DNA, and the delta Rn is maintained at about 0 (figure 3 a); the melting temperature of PMA-qLAMP on the lactobacillus paracasei amplification product is basically the same, and is about 82.4 ℃ (figure 3b), which indicates that the PMA-qLAMP does not have nonspecific amplification reaction, therefore, the specificity of the designed primer is verified, the sensitivity of the primer is high, and the result is accurate.
Example 11 method for detecting Lactobacillus paracasei in Dairy products detection Limit
In this example, the method for detecting lactobacillus paracasei in dairy products in example 4 is adopted, and the detection limit of 10% skim milk powder solution is determined by the following specific determination method:
s1, detection limit of L.paracasei CGMCC4691 in pure culture
Culturing lactobacillus paracasei CGMCC4691 in MRS culture medium at 37 ℃ for 24h, placing lactobacillus paracasei suspension in optimized PMA for treatment, continuously diluting 1mL of viable suspension by 10 times, and counting plates, wherein the results are shown in Table 4;
TABLE 4
Extracting DNA of 1mL of live bacterial cells by a boiling water bath method, dissolving the DNA in 100 mu L of sterile distilled water, detecting the genome DNA of the live strain lactobacillus paracasei diluted by 10 times in series by using PMA-qLAMP, repeating the detection of the DNA at each diluent dose for three times, calculating the average value of three Ct values, and drawing a standard curve (1cycle is 1min) by taking the average value of the Ct values as an ordinate and the logarithm of the number of the live bacterial cells of the lactobacillus paracasei corresponding to a DNA template as an abscissa, wherein the standard curve is shown in figure 4;
as can be seen from FIG. 4, the linear relationship between the Ct value in the range of 8.88 to 25.29min and the logarithm of the viable Lactobacillus paracasei concentration in the range of 8.86 to 2.86 (R2-0.9983) means that PMA-qLAMP can accurately measure the Lactobacillus paracasei concentration in the range of 7.3 × 102CFU/mL～7.3×108Concentration in the CFU/mL range;
s2, detection limit of L.paracasei CGMCC4691 in fermented milk
Detecting by a plate counting method, determining that lactobacillus paracasei does not exist in the fermented milk before artificial treatment, culturing L. paracasei CGMCC4691 in an MRS culture medium at 37 ℃ for 24 hours, taking 3mL of lactobacillus paracasei suspension, centrifuging at 10000rpm for 2min, re-centrifuging the collected bacterial cells with 1mL of sterile water under the same condition to be used as raw bacterial suspension, adding the raw bacterial suspension into 9mL of fermented milk, extracting 1mL of suspension containing viable bacteria from 10mL of fermented milk, continuously diluting by 10 times for plate counting, and treating the suspension by using the optimized PMA condition. And collecting 1mL of live lactobacillus paracasei CGMCC4691 artificially added into the fermented milk by centrifugation. The DNA was extracted by the above boiling water bath method and dissolved in 100. mu.L of sterile distilled water. The active lactobacillus paracasei genomic DNA was detected in 10-fold serial dilutions using PMA-qLAMP. The DNA detection of each diluent dose is repeated for 3 times, the average value of 3 Ct values is calculated, the detection limit of PMA-qLAMP on the viable lactobacillus paracasei CGMCC4691 artificially added into the fermented milk is determined, and the result is shown in figure 5;
as is clear from FIG. 5, it was confirmed that the detection limit of Lactobacillus paracasei in the LAMP-detected fermented milk for the gyrB gene was 7.3X 10 in the same manner as the detection limit of the pure culture2CFU/mL。
EXAMPLE 12 quantitative analysis of Lactobacillus paracasei in fermented milk samples
This example is a quantitative analysis experiment of lactobacillus paracasei in a fermented milk sample, and the specific steps are as follows:
s1, taking 50g of fermented milk, equally dividing into 4 groups, and respectively marking as a group 1, a group 2, a group 3, a group 4 and a group 5;
s2, respectively preserving the group 1, the group 2, the group 3, the group 4 and the group 5 at the temperature of 4 ℃ for 4 days, 7 days, 14 days, 21 days and 28 days;
s3, counting the L.paracasei in each group of fermented milk by adopting a PMA-qLAMP method and a plate counting method;
s4, collecting each set of fermented milk into a sterile centrifuge tube, centrifuging at 10000rpm for 2min, collecting bacterial cells, washing the bacterial cells twice by using normal saline, removing impurities except the bacterial cells in the fermented milk, re-suspending the bacterial cells in the normal saline, counting the cells in 1mL of re-suspended bacterial suspension by using a plate counting method, treating 1mL of the re-suspended bacterial suspension by using PMA, detecting and counting by qLAMP, and analyzing the significance difference of the two methods by using a paired sample t test of SPSS 17.0, wherein the result is shown in Table 5;
TABLE 5
As can be seen from table 5, the difference in concentration values measured by the two methods was not statistically significant (P ═ 0.013>0.01), and it was found that the results of the method of the present invention were not significantly different from those of the plate counting method. In addition, the time required for quantifying one strain of lactobacillus paracasei by using a plate counting method is at least 48 hours, and the time can be shortened to 120min by using the method.
SEQUENCE LISTING
<110> Shijiazhuang Junle Baoru Co Ltd
<120> primer combination and application thereof in detection of lactobacillus paracasei in dairy products
<130> 20211130
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 14
<212> DNA
<213> Artificial sequence
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ggctatgtct acat 14
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gcgatacccg ca 12
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gcaactggcc tagaatgtta cgccaaggca aaatgacg 38
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cgttacaaag ggcttccgga tccatggtcg tct 33
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ttcttcatcg gaatcga 17
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Claims (10)
1. A primer combination is composed of an outer primer, an inner primer and a loop primer, and is characterized in that the primer combination is based ongyrThe primer combination of the B gene comprises an outer primer, an inner primer, a loop primer and sequences thereof, wherein the outer primer, the inner primer and the loop primer are respectively as follows:
outer primer FOP: 5'-GGCTATGTCTACAT-3',
An outer primer BOP: 5'-GCGATACCCGCA-3',
Inner primer FIP: 5'-GCAACTGGCCTAGAATGTTACGCCAAGGCAAAATGACG-3',
The inner primer BIP: 5'-CGTTACAAAGGGCTTCCGGATCCATGGTCGTCT-3',
Loop primer FLP: 5'-TTCTTCATCGGAATCGA-3' and
loop primer BLP: 5'-GAAATGGACGCCAAC-3' are provided.
2. Use of a primer combination according to claim 1 for the detection of lactobacillus paracasei in a dairy product.
3. The application of the primer combination to detection of lactobacillus paracasei in dairy products according to claim 2, wherein the primer combination is used as a primer of a qLAMP reaction system, DNA of the dairy product to be detected after PMA treatment is used as a template of the qLAMP reaction system to perform qLAMP reaction, the fluorescence intensity of the qLAMP reaction system is monitored in real time, if the reaction result is positive, the dairy product to be detected contains lactobacillus paracasei, and if the reaction result is negative, the dairy product to be detected does not contain lactobacillus paracasei.
4. The application of the primer combination for detecting lactobacillus paracasei in dairy products according to claim 3, wherein the DNA of the dairy product to be detected is extracted by the following steps:
s11, adding water into the dairy product to be detected for dissolving/diluting, culturing in an MRS culture medium, and then washing cells with sterile normal saline to obtain a liquid alpha to be detected;
s12, adding a PMA solution into the bacterial suspension alpha to obtain a reaction liquid beta, carrying out photophobic reaction on the reaction liquid beta, incubating under a halogen light source, centrifuging, discarding the supernatant, and taking bacterial cells to wash and suspend to obtain a bacterial suspension gamma;
and S13, carrying out boiling water bath treatment on the bacterial suspension gamma, cooling, centrifuging, and taking supernatant to obtain the DNA of the dairy product to be detected.
5. The use of the primer combination according to claim 4 for detecting Lactobacillus paracasei in dairy products, wherein in step S11, the culturing is performed at 35-40 ℃ for 20-30 h.
6. Use of a primer combination according to claim 4 for the detection of Lactobacillus paracasei in a dairy product, characterized in that in step S12:
the concentration of the PMA solution is 10mg/mL;
the concentration of lactobacillus paracasei in the reaction liquid beta is 5 mug/mL;
the reaction is carried out in a dark place for 3-8 min;
the power of the halogen light source is 400-600 w;
the incubation time is 8-15 min;
the centrifugation is carried out at 10000-15000rpm for 1-3 min.
7. Use of a primer combination according to claim 4 for the detection of Lactobacillus paracasei in a dairy product, characterized in that in step S13:
the boiling water bath treatment lasts for 8-15 min;
the temperature after cooling is 20-30 ℃;
the centrifugation is carried out at 10000-15000rpm for 8-15 min.
8. The use of a primer combination according to any one of claims 2-7 for detecting lactobacillus paracasei in dairy products, wherein the qLAMP reaction system comprises, by volume, 3-4 parts of inner primer FIP, 3-4 parts of inner primer BIP, 0.3-0.8 part of outer primer FOP, 0.3-0.8 part of outer primer BOP, 3-5 parts of loop primer FLP, 3-5 parts of loop primer BLP, and 0.8-1.2 parts of template, and further comprises 2-3 parts of 10 × reaction buffer, 0.8-1.2 parts of magnesium sulfate solution, 1.1-1.8 parts of dNTPs, 0.2-0.5 part of betaine, 0.8-1.2 parts of polymerase, 0.2-0.5 part of 10000 × SYBRgreenI, and 1-1.5 parts of sterile distilled water.
9. The use of a primer combination according to claim 8 for detecting lactobacillus paracasei in dairy products, wherein the reaction buffer is Thermopol reaction buffer;
the concentration of the magnesium sulfate solution is 40-60 mmol/L;
the concentration of the dNTPs is 8-15 mmol/L;
the concentration of the inner primer FIP is 8-15 mmol/L;
the concentration of the inner primer BIP is 8-15 mmol/L;
the concentration of the outer primer FOP is 8-15 mmol/L;
the concentration of the outer primer BOP is 8-15 mmol/L;
the concentration of the loop primer FLP is 8-15 mmol/L;
the concentration of the loop primer BLP is 8-15 mmol/L.
10. Use of a primer combination according to any one of claims 2-7 and 9 for detecting lactobacillus paracasei in dairy products, wherein the qLAMP reaction system is covered with mineral oil; the qLAMP reaction temperature is 60-65 ℃ and the time is 35-45 min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255879A (en) * | 2015-11-13 | 2016-01-20 | 中华人民共和国汕头出入境检验检疫局 | Method for detecting lactobacillus rhamnosus through real-time fluorescent PCR based on DPO primer, primer and kit |
| CN109517912A (en) * | 2018-08-06 | 2019-03-26 | 河北省食品检验研究院（国家果类及农副加工产品质量监督检验中心、河北省食品安全实验室） | A kind of purposes of digital pcr primer and its quantitative detection lactobacillus paracasei content |
| CN112029881A (en) * | 2020-09-15 | 2020-12-04 | 石家庄君乐宝乳业有限公司 | Primer pair for detecting lactobacillus paracasei N1115 and application thereof |
-
2021
- 2021-11-30 CN CN202111448801.0A patent/CN113999925A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255879A (en) * | 2015-11-13 | 2016-01-20 | 中华人民共和国汕头出入境检验检疫局 | Method for detecting lactobacillus rhamnosus through real-time fluorescent PCR based on DPO primer, primer and kit |
| CN109517912A (en) * | 2018-08-06 | 2019-03-26 | 河北省食品检验研究院（国家果类及农副加工产品质量监督检验中心、河北省食品安全实验室） | A kind of purposes of digital pcr primer and its quantitative detection lactobacillus paracasei content |
| CN112029881A (en) * | 2020-09-15 | 2020-12-04 | 石家庄君乐宝乳业有限公司 | Primer pair for detecting lactobacillus paracasei N1115 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| HU 等: "Detection of viable Lacticaseibacillus paracasei in fermented milk using propidium monoazide combined with quantitative loop-mediated isothermal amplification", FEMS MICROBIOLOGY LETTERS, vol. 368, pages 1 - 2 * |
| 王力均 等: "应用PMA-qPCR方法快速准确检测发酵乳制品中副干酪乳杆菌活菌的研究", 中国微生态学杂志, vol. 25, no. 1, pages 3 - 1 * |
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