CN112680535A - Droplet digital PCR detection method for Listeria monocytogenes and application thereof - Google Patents
Droplet digital PCR detection method for Listeria monocytogenes and application thereof Download PDFInfo
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Abstract
The invention provides a droplet digital PCR detection method of Listeria monocytogenes and application thereof, relating to the technical field of biological detection. The invention provides a droplet digital PCR detection method of Listeria monocytogenes, which comprises the steps of firstly extracting DNA of a Listeria monocytogenes standard product, a sample to be detected and a non-target strain respectively, then carrying out droplet digital PCR amplification on the obtained DNA to obtain a reaction solution, and finally detecting and analyzing the reaction solution, wherein a probe of the droplet digital PCR amplification has a nucleotide sequence shown as SEQ ID NO.3, a primer has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer has better specificity on the amplification of the Listeria monocytogenes and can accurately determine the content of the Listeria monocytogenes in food. The detection method provided by the invention is simple, short in period, high in sensitivity, high in precision, good in stability and free of cross reaction with escherichia coli and the like.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a droplet digital PCR detection method for Listeria monocytogenes and application thereof.
Background
The microdroplet digital PCR (ddPCR) is a novel nucleic acid quantitative detection technology, is different from the traditional quantitative PCR (qPCR) technology, adopts an absolute quantitative mode, does not depend on a standard curve and a reference sample, directly detects the copy number of a target sequence, has a detection limit of single copy, and has more excellent sensitivity, specificity and accuracy than the traditional RT-PCR. The microdroplet digital PCR technology has great advantages in the aspects of trace nucleic acid sample detection, rare mutation detection under complex background and identification of small difference of expression quantity.
Listeria monocytogenes (Listeria monocytogenes), abbreviated as Listeria monocytogenes (L.M), belongs to the genus Listeria, and is a pathogenic bacterium of zoonosis. The main diseases caused by the bacteria meningitis include bacterial meningitis, septicemia, myocarditis, osteomyelitis, and even abortion in pregnant women and infection in newborn babies. One of the main characteristics of the listeria monocytogenes is that the listeria monocytogenes can grow at low temperature, can survive for 1 year under the environment of minus 20 ℃, can survive at 0-45 ℃, and can propagate in large quantities at the temperature of 4-6 ℃ in a refrigerator refrigerating chamber, so that the listeria monocytogenes has the name of 'refrigerator killer'. According to the statistics of the Centers for Disease Control and Prevention (CDC), there are about 1600-2000 cases of food poisoning by listeria monocytogenes in the united states each year on average, although the pathogenicity rate of the listeria monocytogenes is not very high, the lethality rate is very high, and the lethality rate for adults is as high as 20% -60%, and for newborns is 54% -90%. Therefore, the detection of the listeria monocytogenes in food is very important for guaranteeing the food safety.
The conventional detection method comprises the traditional biological detection, including an EB (Epstein-Barr) enrichment method and an LB (LB) enrichment method, and through the separation culture, biochemical reaction, hemolytic test, cooperative hemolytic test, animal test and the like of microorganisms, the requirements of experimental equipment are not high, the operability is strong, but the detection period is long, and 6d-7d is needed, so that the conventional detection method cannot meet the requirements of online detection in the food production process and rapid detection before food marketing and consumption; the traditional immunological detection method, such as an ELISA detection method, has the advantages that a commercialized Listeria monocytogenes detection kit produced by Germany Baifa company can be used, the ELISA method is simple to operate, depends on the quality of an antibody, has strong specificity, but has relatively high detection limit, and the detection cost of various food samples by using the ELISA kit is high due to the expensive preparation of a monoclonal antibody; the conventional RT-PCR method has the advantages of high sensitivity, short detection time and capability of quantitative detection, but the quantitative detection depends on a standard curve, the detection limit cannot detect single copy, the operation requirement is high, and the problems of false positive and false negative are easily caused. Therefore, the method for establishing the droplet digital PCR (ddPCR) detection method of the listeria monocytogenes in the food can effectively solve the problems of insufficient sensitivity, low detection precision, long detection period, complex operation and the like of the traditional method in food safety detection.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first objective of the present invention is to provide a digital PCR method for detecting listeria monocytogenes droplets, which has the advantages of simple operation, short detection period, high detection precision and good sensitivity, and can solve at least one of the above problems.
The second objective of the present invention is to provide an application of the droplet digital PCR detection method for listeria monocytogenes in food detection.
In order to solve the technical problems, the following technical scheme is adopted:
in a first aspect, the present invention provides a droplet digital PCR detection method for listeria monocytogenes, comprising the following steps: respectively extracting DNAs of a Listeria monocytogenes standard substance, a sample to be detected and a non-target strain, carrying out microdroplet digital PCR amplification on the obtained DNAs to obtain a reaction solution, and finally detecting and analyzing the reaction solution;
the primer for microdroplet digital PCR amplification has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;
the microdroplet digital PCR amplified probe has a nucleotide sequence shown as SEQ ID NO. 3.
As a further technical solution, the listeria monocytogenes standard comprises a plasmid;
preferably, the plasmid comprises a sequence amplified by the primer from listeria monocytogenes.
As a further aspect, the non-target strain comprises at least one of staphylococcus aureus, bacillus aeruginosa, salmonella or escherichia coli.
As a further technical scheme, the reaction system of the microdroplet digital PCR comprises: 9-11 muL of ddPCR Supermix for Probes, 1.8-2.2 muL of upstream primer, 1.8-2.2 muL of downstream primer, 0.9-1.1 muL of probe, 0.9-1.1 muL of template DNA and 3.6-4.4 muL of double distilled water;
preferably, the reaction system of microdroplet digital PCR comprises: 10 mu L of ddPCR Supermix for Probes, 2 mu L of upstream primer, 2 mu L of downstream primer, 1 mu L of probe, 1 mu L of template DNA and 4 mu L of double distilled water.
As a further technical scheme, the concentration of the upstream primer, the downstream primer and the probe in the reaction system is respectively and independently 90-110 mu mol/L, and preferably 100 mu mol/L.
As a further technical scheme, the reaction conditions of the microdroplet digital PCR are as follows: pre-denaturation: 94-95 ℃, 3-10min, 1 cycle; denaturation: 94-95 ℃, 30-40s, annealing: 50-58 ℃ for 60-80s, and 40 cycles; and (3) finishing the reaction: 4-6 ℃, 4-6min, 88-90 ℃, 4-6min, 1 cycle; the temperature control rate is 1.9-2.1 ℃/s;
preferably, the reaction conditions of the microdroplet digital PCR are: pre-denaturation: 95 ℃, 5min, 1 cycle; denaturation: 95 ℃, 30s, annealing: 55.7 ℃, 60s, 40 cycles; and (3) finishing the reaction: 4 ℃, 5min, 90 ℃, 5min, 1 cycle; the temperature control rate was 2 ℃/s.
As a further technical scheme, two ends of the probe respectively contain a fluorescence quenching group and a fluorescence reporting group.
As a further technical solution, the fluorescent reporter group comprises FAM, CY5 or HEX, preferably FAM.
As a further technical scheme, the fluorescent reporter group comprises BHQ1 or BHQ2, preferably BHQ 1.
In a second aspect, the invention provides an application of a droplet digital PCR detection method for Listeria monocytogenes in food detection.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a droplet digital PCR detection method of Listeria monocytogenes, which comprises the steps of firstly extracting DNA of a Listeria monocytogenes standard product, a sample to be detected and a non-target strain respectively, then carrying out droplet digital PCR amplification on the obtained DNA to obtain a reaction solution, and finally detecting and analyzing the reaction solution, wherein a probe of the droplet digital PCR amplification has a nucleotide sequence shown as SEQ ID NO.3, a primer has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer has better specificity on the amplification of the Listeria monocytogenes and can accurately determine the content of the Listeria monocytogenes in food. The detection method is simple, has short period, can realize batch detection of multiple samples in 1d, has the lowest detection Limit (LOD) of 2.5 copies/mu L and the quantitative Limit (LOQ) of 5.0 copies/mu L, and meets the requirement of rapid quantitative detection; the sensitivity is high, the precision is high, the stability is good, and no cross reaction exists among bacillus aeruginosa, escherichia coli, staphylococcus aureus, salmonella and the like; the detection of the low-concentration infectious bacteria sample is more accurate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a specific experimental scattergram of the droplet digital PCR detection method of Listeria monocytogenes of the present invention;
FIG. 2 is a histogram of the specificity experiment of the droplet digital PCR detection method of Listeria monocytogenes of the present invention;
FIG. 3 is a sensitivity experiment scattergram of the droplet digital PCR detection method of Listeria monocytogenes of the present invention;
FIG. 4 is a linear experimental standard curve diagram of the droplet digital PCR detection method for Listeria monocytogenes of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the present invention provides a droplet digital PCR detection method for listeria monocytogenes, comprising the following steps: respectively extracting DNAs of a Listeria monocytogenes standard substance, a sample to be detected and a non-target strain, carrying out microdroplet digital PCR amplification on the obtained DNAs to obtain a reaction solution, and finally detecting and analyzing the reaction solution;
the primer for microdroplet digital PCR amplification has the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO. 2. In the invention, the hlyA specific gene of the Listeria monocytogenes is used as a detection gene, and the conserved region is selected for microdroplet digital PCR amplification.
The microdroplet digital PCR amplified probe has a nucleotide sequence shown as SEQ ID NO. 3.
The invention provides a droplet digital PCR detection method of Listeria monocytogenes, which comprises the steps of firstly extracting DNAs of Listeria monocytogenes standard products, sample bacteria to be detected and non-target strains respectively, then carrying out droplet digital PCR amplification on the obtained DNAs to obtain reaction liquid, and finally detecting and analyzing the reaction liquid, wherein a probe of the droplet digital PCR amplification has a nucleotide sequence shown as SEQ ID NO.3, a primer has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer has better specificity on the amplification of the Listeria monocytogenes and can accurately determine the content of the Listeria monocytogenes in food. The detection method is simple, has short period, can realize batch detection of multiple samples in 1d, has the lowest detection Limit (LOD) of 2.5 copies/mu L and the quantitative Limit (LOQ) of 5.0 copies/mu L, and meets the requirement of rapid quantitative detection; the sensitivity is high, the precision is high, the stability is good, and no cross reaction exists among bacillus aeruginosa, escherichia coli, staphylococcus aureus, salmonella and the like; the detection of the low-concentration infectious bacteria sample is more accurate.
In some preferred embodiments, the listeria monocytogenes standard includes, but is not limited to, a plasmid;
preferably, the plasmid comprises a sequence amplified by the primer from listeria monocytogenes. In the present invention, listeria monocytogenes standard is used as a positive control group. In the present invention, the phrase "comprising a sequence amplified by the primer of Listeria monocytogenes" refers to a sequence amplified by the primer (SEQ ID NO.1 and SEQ ID NO.2) of Listeria monocytogenes.
In some preferred embodiments, the non-target strains include, but are not limited to, at least one of staphylococcus aureus, bacillus aeruginosa, salmonella or escherichia coli, or other strains known to those of skill in the art. In the invention, the listeria monocytogenes is used as a sample to be detected, the non-target strain is used as a control group, and the detection of the listeria monocytogenes is realized by adopting microdroplet digital PCR.
The term "non-target strain" refers to a strain other than listeria monocytogenes.
In some preferred embodiments, the reaction system of microdroplet digital PCR comprises: 9-11 muL of ddPCR Supermix for Probes, 1.8-2.2 muL of upstream primer, 1.8-2.2 muL of downstream primer, 0.9-1.1 muL of probe, 0.9-1.1 muL of template DNA and 3.6-4.4 muL of double distilled water;
in some preferred embodiments, the reaction system of microdroplet digital PCR comprises: 10 mu L of ddPCR Supermix for Probes, 2 mu L of upstream primer, 2 mu L of downstream primer, 1 mu L of probe, 1 mu L of template DNA and 4 mu L of double distilled water.
In the invention, each component in the reaction system of the microdroplet digital PCR is further optimized and adjusted, so that the PCR reaction is more favorably carried out.
In some preferred embodiments, the concentration of the forward primer, the backward primer and the probe in the reaction system is 90-110. mu. mol/L, and may be, for example, but not limited to, 90. mu. mol/L, 94. mu. mol/L, 98. mu. mol/L, 102. mu. mol/L, 106. mu. mol/L or 110. mu. mol/L, preferably 100. mu. mol/L.
The concentration of the upstream primer, the concentration of the downstream primer and the concentration of the probe in the reaction system are further optimized and adjusted, so that the PCR reaction is more favorably carried out.
In some preferred embodiments, the reaction conditions of the microdroplet digital PCR are: pre-denaturation: 94-95 ℃, 3-10min, 1 cycle; denaturation: 94-95 ℃, 30-40s, annealing: 50-58 ℃ for 60-80s, and 40 cycles; and (3) finishing the reaction: 4-6 ℃, 4-6min, 88-90 ℃, 4-6min, 1 cycle; the temperature control rate is 1.9-2.1 ℃/s;
preferably, the reaction conditions of the microdroplet digital PCR are: pre-denaturation: 95 ℃, 5min, 1 cycle; denaturation: 95 ℃, 30s, annealing: 55.7 ℃, 60s, 40 cycles; and (3) finishing the reaction: 4 ℃, 5min, 90 ℃, 5min, 1 cycle; the temperature control rate was 2 ℃/s.
By further optimizing and adjusting the reaction conditions of the microdroplet digital PCR, the method is favorable for the PCR reaction and shortens the detection period.
In some preferred embodiments, the two ends of the probe respectively contain a fluorescence quenching group and a fluorescence reporter group. In this embodiment, the type of the fluorescence reporter group and the fluorescence quencher group contained in the probe is not particularly limited, and any group that can be linked to the probe and can perform a corresponding characterization function may be used.
In some preferred embodiments, the fluorescent reporter includes, but is not limited to FAM, CY5, or HEX, preferably FAM.
In some preferred embodiments, the fluorescent reporter comprises BHQ1 or BHQ2, preferably BHQ 1.
In a second aspect, the invention provides an application of a droplet digital PCR detection method for Listeria monocytogenes in food detection.
The provided method for detecting the listeria monocytogenes by the droplet digital PCR can realize the detection of the listeria monocytogenes, and does not limit the source of the listeria monocytogenes, so that the method can be applied to the detection of the listeria monocytogenes in food.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Example 1 method for microdroplet digital PCR (ddPCR) detection of Listeria monocytogenes
1 preparing Listeria monocytogenes standard substance
1.1 Listeria monocytogenes conserved sequence amplification
An amplification primer:
PrimeF：5’-TTAATTTCATCCATGGCACCAC-3’(SEQ ID NO.1)；
PrimeR：5’-ACTTATCGATTTCATCCGCGTG-3’(SEQ ID NO.2)；
the PCR amplification system (the experiment was performed using the PrimeStar high fidelity enzyme kit from TAKARA, Japan) is shown in Table 1 below:
TABLE 1 PCR amplification System
The DNA amplification procedure conditions are shown in table 2 below:
TABLE 2 DNA amplification reaction procedure
1.2 purification and recovery of conserved sequence amplification products
The conservative sequence PCR amplification product is purified and recovered by adopting a gel cutting recovery mode (the experiment adopts an agarose gel purification kit of Beijing Tiangen Biotechnology Co., Ltd.), and the operation steps are as follows:
a: loading 20 μ L/well of PCR product into 2% agarose gel electrophoresis, and performing electrophoresis for 30 min; placing the gel under a gel imager for ultraviolet irradiation, cutting off a gel block with a target strip, transferring the gel block into a sterilized centrifuge tube, and weighing the weight of the gel block;
b: adding 3 times volume of PN sol solution into the gel block, wherein the gel weight is calculated according to the volume of 100 mu L per 0.1g, adding 300 mu L of PN sol solution, and placing the solution in a metal bath at 50 ℃ for complete dissolution;
c: cooling the solution obtained in the previous step to room temperature, transferring the solution to an adsorption column provided with a collecting pipe, and centrifuging the solution for 1min at 13000 r/min;
d: adding 700 μ L of rinsing solution PW (containing anhydrous ethanol) into the adsorption column, and centrifuging at 13000r/min for 1 min;
e: repeating the step d, centrifuging at 13000r/min for 2min, and removing the rinsing liquid as much as possible; drying the adsorption column to prevent the influence of the residual rinsing liquid on the next experiment;
f: and (3) suspending and dropwise adding 65-70 mu L of preheated double distilled water onto the adsorption film, standing for 2min, centrifuging at 13000r/min for 1min, and collecting a DNA solution, namely a PCR purified product.
1.3 construction of Standard recombinant plasmid
Ligation system for ligation (5 minTA/Blunt-Zero Cloning Kit from Nanjing Novodka Biotechnology Ltd.) is shown in Table 3:
TABLE 3 connection System
| Components | Volume of |
| 5×TA/Blunt-Zero Cloning Mix | 1μL |
| PCR purified product | 1μL |
| Double distilled water | 3μL |
And (3) centrifuging the connection system at a low speed, collecting all liquid, namely connection products, reacting for 5min at 37 ℃ in a metal bath, and placing the centrifugal tube on ice to prepare for conversion.
And (3) conversion reaction:
a: 2.5. mu.L of the ligation product was transformed into 50. mu.L of DH 5. alpha. competent cells, mixed well and ice-cooled for 30 min;
b: after the ice bath is finished, transferring the mixture into a metal bath, performing heat shock for 45s at 42 ℃, and then quickly transferring the mixture into the ice bath for 2 min;
c: adding 500 mu L of sterile antibiotic-free SOC culture medium into a centrifuge tube, reversing, uniformly mixing, placing in a constant temperature shaking table at 37 ℃ and 200r/min, and incubating for 1h to recover bacteria;
d: centrifuging the recovered bacteria at 800r/min for 30s, discarding the supernatant, uniformly coating the bacteria liquid in a flat LB culture medium containing kanamycin, and inverting the flat LB culture medium to culture at 37 ℃ for 12 h.
Colony PCR identification, selecting a single clone to 10 mu L LB liquid culture medium and mixing uniformly as a bacterial liquid template, wherein a specific PCR identification system (adopted in experiments) is shown in a table 4:
TABLE 4PCR identification System
The DNA amplification reaction procedure is shown in table 5 below:
TABLE 5 DNA amplification reaction procedure
| Procedure | Temperature (. degree.C.) | Time |
| Pre-denaturation | 95 | 3min |
| Denaturation of the material | 95 | 15s |
| Annealing | 55 | 15s |
| Extension of | 72 | 85 |
And (4) observing the PCR product after electrophoresis, selecting positive clones, performing enzyme digestion verification and sequencing.
The copy number of the standard was measured by first extracting the plasmid, and measuring the concentration of the plasmid solution with a NanoDrop accountant (Thermofeisher, USA) according to the formula:
copy number (copies/. mu.L) ═ 6.02X 1023×(ng/μL×10-9)/DNA length×660
The copy number of the standard sample was calculated to be 5.0X 1010copies/μL。
2 enrichment culture of non-target strains
Culturing the non-target strains in LB culture solution at 37 ℃ for 16-24h for enrichment to obtain enrichment solution of a control group;
the non-target strains are as follows: staphylococcus aureus, bacillus aeruginosa, salmonella and escherichia coli.
3 extraction of DNA
3.1 extraction of template DNA
a: placing 5ml of the bacteria culture solution into a centrifuge tube, centrifuging for 1min at 10000r/min, and removing the supernatant;
b: dissolving lysozyme (concentration: 0.03g/mL) in PBS buffer solution, re-dissolving thallus with lysozyme solution, and performing enzymolysis at 37 deg.C for 1.5 h;
c: adding 20 μ L of protease K solution into the tube, mixing, adding 220 μ L of buffer GB, shaking for 15s, standing at 70 deg.C for 10 min;
d: adding 220 microliter of absolute ethyl alcohol when the solution becomes clear, fully oscillating and uniformly mixing for 15s, and generating flocculent precipitates;
e: transferring the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 with a collecting pipe, and centrifuging at 12000r/min for 30 s;
f: adding 500 μ L buffer GD (containing absolute ethanol) into adsorption column CB3, and centrifuging at 12000r/min for 30 s; adding 600 μ L of rinsing solution PW (containing anhydrous ethanol) into adsorption column CB3, and centrifuging at 12000r/min for 30 s;
g: repeating the operation of the step f, and centrifuging at 12000r/min for 2 min; the adsorption column CB3 was dried to ensure that there was no residual rinse.
h: and suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption membrane, standing for 5min, centrifuging at 12000r/min for 2min, and collecting the centrifugate, namely the extracted template DNA.
3.2 extraction of DNA from samples to be tested
a. Taking 1mL of a sample to be detected, inoculating the sample to 5mL of the culture medium of the enrichment broth of Lee, uniformly shaking, and culturing in an incubator at 37 ℃ for 12 h;
b. placing 5ml of the bacteria culture solution into a centrifuge tube, centrifuging for 1min at 10000r/min, and removing the supernatant;
dissolving lysozyme (concentration: 0.03g/mL) in PBS buffer solution, re-dissolving the thallus with lysozyme solution, and performing enzymolysis for 1.5h at 37 ℃;
d. adding 20 μ L of protease K solution into the tube, mixing, adding 220 μ L of buffer GB, shaking for 15s, standing at 70 deg.C for 10 min;
e. adding 220 microliter of absolute ethyl alcohol when the solution becomes clear, fully oscillating and uniformly mixing for 15s, and generating flocculent precipitates;
f. transferring the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 with a collecting pipe, and centrifuging at 12000r/min for 30 s;
g. adding 500 μ L buffer GD (containing absolute ethanol) into adsorption column CB3, and centrifuging at 12000r/min for 30 s; adding 600 μ L of rinsing solution PW (containing anhydrous ethanol) into adsorption column CB3, and centrifuging at 12000r/min for 30 s;
h. repeating the operation of the step f, and centrifuging at 12000r/min for 2 min; the adsorption column CB3 was dried to ensure that there was no residual rinse.
i. And suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption membrane, standing for 5min, centrifuging at 12000r/min for 2min, and collecting the centrifugate, namely the extracted template DNA.
4ddPCR amplification reaction
Amplification primers and probes:
PrimeF：5’-TTAATTTCATCCATGGCACCAC-3’(SEQ ID NO.1)；
PrimeR：5’-ACTTATCGATTTCATCCGCGTG-3’(SEQ ID NO.2)；
and (3) probe: 5 '-FAM-TCCGCCTGCAAGTCCTAAGACGCC-BHQ 1-3' (SEQ ID NO. 3).
The ddPCR amplification system (ddPCR Supermix (for probes, No dUTP) kit from the company Bio-Rad, USA) as shown in the following Table 6 was obtained:
TABLE 6 ddPCR amplification System
| Components | Volume of |
| ddPCR Supermix for Probes | 10μL |
| PrimeF (concentration: 100. mu. mol/L) | 2μL |
| PrimeR (concentration: 100. mu. mol/L) | 2μL |
| Probe (concentration: 100. mu. mol/L) | 1μL |
| Template DNA | 1μL |
| Double distilled water | 4μL |
Add 20. mu.L of the reaction to a microtiter plate (for probes) using the Bio-Rad Droplet Generation Oil kit, U.S.A.) and add 70. mu.L of the Generation Oil to each well; generating microdroplets from 20 mu L of reaction system mixture by using a microdroplet generator, and amplifying by using a PCR instrument;
the ddPCR amplification reaction program is as follows: 95 ℃/5 min; 95 ℃/30s, 55.7 ℃/1min, 40 cycles; 4 ℃/5min, 90 ℃/5 min; the temperature control rate is 2 ℃/s; after the system is set up, the digital PCR reaction is carried out. The fluorescence analysis step employs FAM single fluorescence channel.
5 detection method
5.1 specificity experiments for ddPCR amplification
The DNA of Listeria monocytogenes, Staphylococcus aureus, Bacillus aeruginosa, Salmonella and Escherichia coli are respectively used as templates to carry out ddPCR detection, and the specificity is verified, and the result is shown in figure 1 and figure 2. In FIG. 1, the number of detected droplets is plotted on the abscissa, the intensity of detected fluorescence is plotted on the ordinate, the gray-black part is determined as a negative droplet, the blue part is determined as a positive droplet, wherein the positive droplet and the negative droplet are clearly divided into two clusters, and the number of droplets dispersed in the middle is small, indicating that the DNA amplification effect is good; in FIG. 2, the abscissa represents the detected fluorescence intensity, the ordinate represents the number of detected droplets, one peak with low fluorescence intensity on the left side of the histogram is a negative droplet, the other peak with high fluorescence signal intensity on the right side is a positive droplet, the positive peak and the negative peak are separated obviously, and no interference exists in the middle, which indicates that the ddPCR primer probe and the amplification system are suitable for quantitative analysis of Listeria monocytogenes.
5.2 sensitivity assay for ddPCR amplification
Sequentially diluting the listeria monocytogenes standard recombinant plasmids according to 10-fold gradient concentration, wherein the concentration gradient range is as follows: 5.0X 105copies/μL～5.0×100Samples of copies/. mu.L were subjected to ddPCR with three replicates per gradient set-up and the results are shown in FIG. 3 with a minimum detection Limit (LOD) of 2.5 copies/. mu.L and a limit of quantitation (LOQ) of 5.0 copies/. mu.L.
5.3 Linear experiment of ddPCR amplification
Sequentially diluting the recombinant plasmid of the Listeria monocytogenes standard product according to 10-fold gradient concentration to obtain final concentrations of 5.0 × 1010copies/μL～5.0×100Samples of copies/. mu.L were subjected to ddPCR with three replicates per gradient, the results of the standard curve are shown in FIG. 4, at concentrations higher than 5.0X 105The samples of copies/. mu.L did not allow accurate quantitation of ddPCR. The detection method provided by the invention has the advantages of high sensitivity, high precision and good stability, and the SD and CV are respectively 0.23 and 3.62% at 5.0 copies/mu L; the linear relation of multiple dilution is good, and the correlation coefficient (r)>0.990。
6. Amplification product specificity assay
Reaction system:
dissolution curve experimental procedure:
the qPCR program was: pre-denaturation at 95 ℃ for 30 s; the reaction is circulated for 40 cycles of reaction at 95 ℃ for 10s and at 60 ℃ for 30 s; dissolution curve: 95 ℃ for 10s, 60 ℃ for 60s and 95 ℃ for 15 s.
The result shows that the primer provided by the invention has an obvious single peak and good primer specificity.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Beijing college of agriculture
Droplet digital PCR (polymerase chain reaction) detection method of <120> listeria monocytogenes and application thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence
<400> 1
ttaatttcat ccatggcacc ac 22
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<400> 2
acttatcgat ttcatccgcg tg 22
<210> 3
<211> 24
<212> DNA
<213> Artificial sequence
<400> 3
tccgcctgca agtcctaaga cgcc 24
Claims (10)
1. A droplet digital PCR detection method for Listeria monocytogenes, which is characterized by comprising the following steps: respectively extracting DNAs of a Listeria monocytogenes standard substance, a sample to be detected and a non-target strain, carrying out microdroplet digital PCR amplification on the obtained DNAs to obtain a reaction solution, and finally detecting and analyzing the reaction solution;
the primer for microdroplet digital PCR amplification has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;
the microdroplet digital PCR amplified probe has a nucleotide sequence shown as SEQ ID NO. 3.
2. The method for droplet digital PCR detection of listeria monocytogenes of claim 1, wherein the listeria monocytogenes standard comprises a plasmid;
preferably, the plasmid comprises a sequence amplified by the primer from listeria monocytogenes.
3. The method for droplet digital PCR detection of listeria monocytogenes of claim 1, wherein the non-target strain comprises at least one of staphylococcus aureus, bacillus aeruginosa, salmonella or escherichia coli.
4. The method of claim 1, wherein the droplet-based digital PCR reaction system comprises: 9-11 muL of ddPCR Supermix for Probes, 1.8-2.2 muL of upstream primer, 1.8-2.2 muL of downstream primer, 0.9-1.1 muL of probe, 0.9-1.1 muL of template DNA and 3.6-4.4 muL of double distilled water;
preferably, the reaction system of microdroplet digital PCR comprises: 10 mu L of ddPCR Supermix for Probes, 2 mu L of upstream primer, 2 mu L of downstream primer, 1 mu L of probe, 1 mu L of template DNA and 4 mu L of double distilled water.
5. The method for the microdroplet digital PCR detection of Listeria monocytogenes of claim 4, wherein the concentration of the upstream primer, the downstream primer and the probe in the reaction system is 90-110 μmol/L, preferably 100 μmol/L.
6. The method of claim 1, wherein the conditions for performing the microdroplet digital PCR are as follows: pre-denaturation: 94-95 ℃, 3-10min, 1 cycle; denaturation: 94-95 ℃, 30-40s, annealing: 50-58 ℃ for 60-80s, and 40 cycles; and (3) finishing the reaction: 4-6 ℃, 4-6min, 88-90 ℃, 4-6min, 1 cycle; the temperature control rate is 1.9-2.1 ℃/s;
preferably, the reaction conditions of the microdroplet digital PCR are: pre-denaturation: 95 ℃, 5min, 1 cycle; denaturation: 95 ℃, 30s, annealing: 55.7 ℃, 60s, 40 cycles; and (3) finishing the reaction: 4 ℃, 5min, 90 ℃, 5min, 1 cycle; the temperature control rate was 2 ℃/s.
7. The method of claim 1, wherein said probe comprises a fluorescence quencher and a fluorescence reporter at each end.
8. The method for droplet digital PCR detection of listeria monocytogenes of claim 7, wherein the fluorescent reporter comprises FAM, CY5, or HEX, preferably FAM.
9. The method for the microdroplet digital PCR detection of Listeria monocytogenes of claim 7, wherein the fluorescent reporter comprises BHQ1 or BHQ2, preferably BHQ 1.
10. Use of the method for droplet digital PCR detection of listeria monocytogenes of any one of claims 1-9 for food detection.
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Non-Patent Citations (3)
| Title |
|---|
| ANNA KRISTINA WITTE等: "Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR", 《ANALYTICAL AND BIOANALYTICAL CHEMISTRY》 * |
| 程逸宇等: "微滴数字PCR定量检测乳制品中单核细胞增生李斯特氏菌", 《中国乳品工业》 * |
| 赵丽青等: "数字PCR定量检测食品中单核细胞增生李斯特氏菌方法的研究", 《食品安全质量检测学报》 * |
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