CN112980972B - Method for quantitatively detecting quail-derived ingredients based on ddPCR technology - Google Patents
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Abstract
The invention discloses a method for quantitatively detecting quail-derived components based on a ddPCR technology, belonging to the field of molecular biological analysis. The method comprises the steps of removing moisture of a sample to be detected through vacuum freeze drying, grinding the sample into powder to obtain a powder sample which is dried uniformly, extracting DNA of the powder sample to be used as a template, adding a ddPCR primer pair and a probe of quail into an amplification reaction system, generating microdroplets by using a microdroplet generator, carrying out amplification by using a ddPCR instrument, reading signals by using the ddPCR instrument after the amplification is finished, analyzing data, and obtaining a quantitative detection result by contrasting a standard curve. According to the invention, the sample freeze-drying treatment method can improve the sample uniformity and simultaneously remove the interference of the moisture content on the quail-derived component quantification; the ddPCR method has high specificity and sensitivity, the limit of quantification is 5 mu g/mg, the limit of detection is 1 mu g/mg, and the ddPCR method can meet the quantitative detection work of quail-derived ingredients in complex samples.
Description
Technical Field
The invention relates to a method for quantitatively detecting quail-derived ingredients based on a ddPCR technology, belonging to the field of molecular biological analysis.
Background
Quails, also known as quails and wild geese, are one of the common poultry raising in China. The quail meat has delicious taste and high nutritive value, is used as high-quality complete protein, has low fat rate, is rich in various amino acids necessary for the growth and development of human bodies, and is easy to digest and absorb by the human bodies. Quail is known as animal ginseng, and the Li Shizhen Ben gang mu points out that quail meat and eggs have the efficacies of tonifying qi in the middle-jiao, tonifying five internal organs, forming muscles and bones, resisting cold and summer heat and removing heat accumulation, so the quail is popular with consumers. The phenomenon that peeled chicken, sparrows or young pigeons are used to serve as quail meat is sometimes generated in the market, and the adulteration event is often prohibited. The current standard method for identifying various animal sources is generally based on a common PCR technology or a fluorescent PCR technology, only qualitative detection can be carried out on target animal source components in a sample, and accurate quantification cannot be carried out, so that the limitation of the technology forms a certain obstacle to market supervision. Therefore, research on new technologies for detecting animal sources in food and development of new methods for quantitatively detecting animal source components are urgent.
The detection technology of the Droplet digital PCR (ddPCR) is a novel nucleic acid quantitative detection technology. The ddPCR principle is that a PCR reaction system is micro-titrated before PCR reaction is carried out, mixed template DNA is dispersed in about twenty thousand micro-droplets, only 1 template DNA molecule exists in most micro-droplets, and a fluorescence signal is read after the PCR amplification program is finished, so that the number of the template DNA molecules in the system is counted. The method can realize quantitative analysis on the target DNA, can realize absolute quantification of the DNA copy number of a sample with low DNA content concentration, and has high sensitivity and accuracy. At present, the application research aiming at the ddPCR technology mainly focuses on the fields of pathogen detection, transgenic food detection and the like, and animal-derived quantitative identification in food is just started.
Disclosure of Invention
The invention aims to provide a method for detecting quail-derived component content based on ddPCR, which adopts a vacuum freeze-drying method to process samples to reduce the interference of moisture content on quail-derived component quantification and simultaneously improves the sample uniformity; by using the specific fluorescent probe and the primer pair and constructing the linear relation between the concentration of the quail-derived substance and the copy number of the target DNA, the one-step conversion between the actual quality of the quail-derived component and the copy number of the target DNA is realized, the quantitative process is simplified, and the quantitative detection of the quail-derived component is more accurate and faster.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a method for detecting quail-derived component content based on ddPCR, which comprises the following steps:
(1) pretreating a sample to be detected, diluting the sample to be detected to 5-1000 mu g/mg, and extracting DNA;
(2) detecting the copy number of the DNA in the step (1) by ddPCR;
(3) determining the content of the quail meat-derived component substances in the sample to be detected in the step (1) according to a standard curve;
the reaction system of the ddPCR in the step (2) contains an upstream primer, a downstream primer and a probe for amplifying quail source, and the sequences of the upstream primer, the downstream primer and the probe are as follows:
and (3) probe: 5'-AGCAGCCAACACAGCACCCA-3', respectively;
an upstream primer: 5'-ACAGCAACGTTCTTCAGCTC-3', respectively;
a downstream primer: 5'-CTGGGAGCGCTTGCTTTATT-3', respectively;
the 5 'end of the probe is modified by FAM, and the 3' end of the probe is modified by BHQ 1; wherein FAM represents a fluorescent reporter group, and BHQ1 represents a quencher group.
In one embodiment, the standard curve in step (3) of the above method is prepared by: pretreating a quail meat sample, weighing the mass of the quail meat sample, extracting DNA in the quail meat sample, determining the copy number of the DNA by ddPCR, and establishing a linear relation between the content of quail-derived component substances and the copy number of the DNA to obtain a standard curve.
In one embodiment, the specific steps for preparing the standard curve are as follows: adding 10 mg of quail meat dry powder into 200 mu L of lysis solution (contained in the DNA extraction kit) to obtain mixed solution, diluting the mixed solution by using the lysis solution according to a proportion to obtain diluted solutions with the contents of 1000, 500, 200, 100, 50, 10, 5 and 1 mu g/mg, adding 20 mu L of protease K into each diluted solution, digesting at a constant temperature of 60 ℃ for 2 hours, and extracting DNA.
In one embodiment, the standard curve is Gn = 2.85C + 37.915, where Gn is the copy number of DNA of the sample and C is the quail-derived ingredient content.
In one embodiment, the pretreatment is to divide a quail meat sample or a sample to be detected into small samples of about 1 cm × 1 cm × 1 cm, and dry the samples for 48-72 hours at a vacuum degree of 0.090-0.100 mbar and a temperature of-60 to-55 ℃ by using a vacuum freeze drying technology; and grinding the freeze-dried sample into a powdery sample by using a tissue grinder to ensure the uniformity of the sample.
In one embodiment, the reaction system of ddPCR is 20 μ L in total, and comprises: ddPCR Super mix for Probes 10. mu.L, probe 0.5. mu.L, forward primer and reverse primer 1.8. mu.L each, DNA 1. mu.L, ddH2Make up to 20. mu.L of O.
In one embodiment, the initial concentration of the forward primer, the reverse primer and the probe is 10. mu. mol/L.
In one embodiment, the reaction system of the ddPCR is 20 μ L, comprising: mu.L of DNA extract was used as template, and the forward primer and the reverse primer at a final concentration of 900 nmol/L, the probe at a final concentration of 250 nmol/L, and 10. mu.L of ddPCR Super mix for Probes, ddH were added2Make up to 20. mu.L of O.
In one embodiment, the amplification procedure of ddPCR is: 10 min at 95 ℃, 30 s at 94 ℃, 1 min at 60 ℃, 40 cycles, and 10 min at 98 ℃.
The invention also provides a quail-derived detection kit, which comprises the following reagents: an upstream primer, a downstream primer and a probe for amplifying quail-derived components.
In one embodiment, the sequences of the forward primer, the reverse primer and the probe are as follows:
and (3) probe: 5'-AGCAGCCAACACAGCACCCA-3', respectively;
an upstream primer: 5'-ACAGCAACGTTCTTCAGCTC-3', respectively;
a downstream primer: 5'-CTGGGAGCGCTTGCTTTATT-3' are provided.
In one embodiment, the 5 'end of the probe is modified with FAM and the 3' end is modified with BHQ 1; wherein FAM represents a fluorescent reporter group, and BHQ1 represents a quencher group.
In one embodiment, the reaction system of the detection kit is as follows: 1 μ L of DNA extract was used as template, and the forward primer, the reverse primer and the probe and 10 μ L of ddPCR Super mix for Probes, ddH were added2Make up to 20. mu.L of O.
In one embodiment, the reaction system of the detection kit is as follows: ddPCR Super mix for Probes10. mu.L of 10. mu. mol/L probe 0.5. mu.L, 10. mu. mol/L forward primer and reverse primer 1.8. mu.L each, template DNA 1. mu.L, ddH2Make up to 20. mu.L of O.
In one embodiment, the amplification procedure of the detection kit on the droplet digital PCR is as follows: 10 min at 95 ℃, 30 s at 94 ℃, 1 min at 60 ℃, 40 cycles, and 10 min at 98 ℃.
Compared with the prior art, the invention has the following beneficial effects:
the quail-derived component in the complex component sample can be quantitatively detected by utilizing the method to detect the quail-derived component and carrying out sample pretreatment, the method has good repeatability and stability, the actual quality of the quail-derived component in the sample can be directly obtained by contrasting a standard curve with the copy number of the target DNA, no internal reference or contrast gene amplification is needed, and the determination process is convenient and quick. The designed ddPCR primer and the probe have better specificity and sensitivity, and the limit of quantification and the limit of detection are respectively 5 mu g/mg and 1 mu g/mg.
Drawings
FIG. 1 shows the results of specific detection.
FIG. 2 is a linear relationship between sample content (. mu.g/mg) and copy number of target DNA (copies/. mu.L).
FIG. 3 is a graph showing the result of ddPCR amplification of quail-derived components in a sample.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1: design of primer probes
The primers and probes were synthesized by Nanjing Kinshire.
Aiming at the design of a single copy gene in a quail cell nuclear genome, specific probe and primer sequences are as follows:
and (3) probe: 5'-AGCAGCCAACACAGCACCCA-3', SEQ ID NO. 1;
an upstream primer: 5'-ACAGCAACGTTCTTCAGCTC-3', SEQ ID NO. 2;
a downstream primer: 5'-CTGGGAGCGCTTGCTTTATT-3', SEQ ID NO. 3;
wherein the 5 'end of the probe is modified by FAM, and the 3' end is modified by BHQ 1; wherein FAM represents a fluorescent reporter group, and BHQ1 represents a quencher group.
The invention adopts a fluorescent probe method, and the detection principle is to utilize a fluorescent labeled specific probe to identify a template. Compared with SYBR dye method in the prior art, the fluorescence labeling specific probe has stronger specificity and lower background interference.
Example 2: specificity verification of primer probes
(1) Sample preparation: quail meat, chicken, duck, goose, pigeon, mutton, beef and pork samples were from the local farmer market in Nanjing.
(2) Reagents and instruments: the DNA extraction kit was purchased from Tiangen Biotechnology (Beijing) Ltd. Premix Ex Taq (Takara, Japan); U.S. Bio-Rad and CFX96 Touch fluorescent PCR instruments.
(3) And (3) specificity verification: the DNA of quail meat, chicken, duck, goose, mutton, beef and pork was extracted as template, QPCR amplification was performed using the primer probes of example 1, three replicates per sample, and ddH was used simultaneously2O replaced DNA template as blank control.
An amplification reaction system: premix Ex Taq 12.5. mu.L, upstream and downstream primers 0.5. mu.L each, Probe 1. mu.L, template 1. mu.L, ddH2O 9.5 μL。
The reaction conditions are as follows: 5 min at 95 ℃; 95 ℃ for 15 s, 60 ℃ for 1 min, 40 cycles.
The results are shown in FIG. 1, the amplification curves in the reaction system containing the quail DNA template are obvious, the repeatability of the three amplification curves is better, and the reaction system containing other sample DNA templates and the blank control group have no amplification curve. The quail-derived primer and the probe designed by the invention have specificity and better reproducibility, and do not have cross reaction with DNA of other common meat.
Example 3: quail-derived ddPCR quantitative detection standard curve design and linear range verification
(1) Sample preparation: quail meat samples were from the local farmer market in Nanjing. The quail meat sample is divided into small samples of about 1 cm multiplied by 1 cm, and the samples are dried for 48-72 hours at the temperature of minus 60 ℃ under the vacuum degree of less than 0.100 mbar by using the vacuum freeze drying technology, so that the influence of the moisture content on the concentration of the quail-derived components in the samples is reduced. The freeze-dried sample is polished into a powdery sample by a tissue grinder, so that the uniformity of the sample is ensured, and the error caused by the non-uniformity of the sample on the quantitative result is avoided.
(2) Reagents and instruments: the DNA extraction kit was purchased from Tiangen Biotechnology (Beijing) Ltd. ddPCR amplification premix ddPCR Super mix for Probes (Bio-Rad, USA); QX200 droplet digital PCR system and CFX96 Touch fluorescent PCR instrument from Bio-Rad, USA.
(3) Quail-derived concentration gradient: 10 mg of quail meat dry powder is added with 200 mu L of GA lysate (contained in a DNA extraction kit) to obtain a mixed solution, the mixed solution is diluted by using the lysate according to a proportion to obtain diluents with the quail meat content of 1000, 500, 200, 100, 50, 10, 5 and 1 mu g/mg, 20 mu L of protease K is respectively added, and the quail meat is digested at a constant temperature of 60 ℃ for 2 hours to extract DNA.
(4) An amplification reaction system and an amplification program: the primer probe in example 1 is used for ddPCR amplification, and the amplification reaction system is as follows: 1.8. mu.L of each of the upstream and downstream primers (final concentration 900 nmol/L), 0.5. mu.L of the probe (final concentration 250 nmol/L), 10. mu.L of ddPCR Super mix for Probes, 1. mu.L of the DNA template, and ddH2O to 20. mu.L.
Droplets were generated using a droplet generator and amplified using a ddPCR instrument.
The ddPCR reaction conditions are as follows: at 95 deg.C for 10 min, at 94 deg.C for 30 s, at 60 deg.C for 1 min, for 40 cycles, at 98 deg.C for 10 min, and storing at 4 deg.C.
ddPCR reactions were performed with three replicates per gradient. The ddPCR instrument normally generates droplets, and the number of droplets generated by all reactions is more than 10000.
TABLE 1 Linear Range
| Quail derived component content (μ g/mg) | Copy number of target DNA (copies/. mu.L) | RDS（%） |
| 1000 | 2885.00±74.08 | 2.57% |
| 500 | 1427.67±24.50 | 1.72% |
| 200 | 694.00±36.59 | 5.27% |
| 100 | 358.33±22.01 | 6.14% |
| 50 | 181.33±17.21 | 9.49% |
| 10 | 56.33±9.45 | 16.78% |
| 5 | 14.33±2.08 | 14.52% |
| 1 | 4.33±1.52 | 35.25% |
The number of positive droplets generated by ddPCR decreased with decreasing quail-derived content (FIG. 3). The statistical result shows that the quail-derived concentration gradient is within the range of 5-1000 mug/mg, the quail-derived substance content and the target DNA copy number have a linear relation, the quail-derived substance concentration is taken as a horizontal coordinate, the average value of the target DNA copy number of each concentration point is taken as a vertical coordinate, and a standard curve is drawn: gn = 2.85C + 37.915, wherein Gn is the copy number of target DNA of the sample, C is the quail-derived component content, and the correlation coefficient (R)2) Is 0.9982, meets the requirement of quantitative detection, and is shown in figure 2.
When the quail source content in the sample is higher, the relative standard deviation is small, and the quantitative result is stable; as the quail-derived concentration decreases, the relative standard deviation becomes larger, and quantification becomes difficult.
And combining with relevant standard regulation, the detection rate is the lowest detection limit when the detection rate is more than or equal to 95 percent, and the relative standard deviation RSD is less than or equal to 25 percent, so that the detection rate is the quantitative lower limit. The quantitative lower limit and the lowest detection limit of the method are respectively 5 mug/mg and 1 mug/mg.
Example 4: artificial mixed series sample detection
(1) Sample preparation: the quail meat and chicken samples are from local farmer markets of Nanjing.
(2) Reagents and instruments: the DNA extraction kit was purchased from Tiangen Biotechnology (Beijing) Ltd. ddPCR amplification premix ddPCR Super mix for Probes (Bio-Rad, USA); QX200 droplet digital PCR system and CFX96 Touch fluorescent PCR instrument from Bio-Rad, USA.
(3) Manual mixing of sample series: adding different amounts of quail meat samples into the chicken samples respectively to prepare mixed samples of which the quail meat proportion is 80%, 50%, 20%, 10% and 1%, wherein each mixed sample is 50 g, vacuum freeze-drying is carried out to prepare uniform powder, 10 mg of the mixed samples are respectively taken to extract DNA for ddPCR detection, and the content of the quail meat in the mixed samples is calculated according to the standard curve obtained in the embodiment 3.
(4) An amplification reaction system and an amplification program: application implementationThe primer probe in example 1 is subjected to ddPCR amplification, and the amplification reaction system is as follows: mu.L (900 nmol/L) of each of the upstream and downstream primers, 0.5. mu.L (250 nmol/L) of the probe, 10. mu.L of ddPCR Super mix for Probes, 1. mu.L of DNA template, and ddH2O to 20. mu.L.
Droplets were generated using a droplet generator and amplified using a ddPCR instrument.
The ddPCR reaction conditions are as follows: at 95 deg.C for 10 min, at 94 deg.C for 30 s, at 60 deg.C for 1 min, for 40 cycles, at 98 deg.C for 10 min, and storing at 4 deg.C.
TABLE 2 proportion of quail meat in chicken meat
| Actual mass fraction (%) | Determination of Mass fraction (%) | RSD（%） | Recovery (%) |
| 80 | 78.22 | 5.67 | 97.78 |
| 50 | 52.03 | 7.22 | 104.06 |
| 20 | 18.77 | 10.25 | 93.85 |
| 10 | 9.54 | 9.78 | 95.40 |
| 1 | 0.82 | 15.89 | 82.00 |
The test results of the artificial adulteration mixing series samples show that the RSD values of three repeated experiments are less than 25 percent and are suitable for quantitative detection on 5 mixing samples with known mass fractions. The ddPCR detection method has higher accuracy under the condition that the content of the quail-derived components in a sample to be detected is higher than 1%, and compared with the actual mass fraction, the recovery rate of the detection result is kept between 82% and 104.06%, so that the requirement of quantitative detection of the quail-derived components in a mixed sample can be met.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> Nanjing City food and drug supervision and inspection institute
<120> method for quantitatively detecting quail-derived ingredients based on ddPCR technology
<130> BAA210356A
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
acagcaacgt tcttcagctc 20
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<400> 3
Claims (10)
1. A method for detecting the content of quail-derived ingredients based on ddPCR is characterized by comprising the following steps: (1) pretreating a sample to be detected, diluting the sample to be detected to 5-1000 mu g/mg, and extracting DNA;
(2) detecting the copy number of the DNA in the step (1) by ddPCR;
(3) determining the content of the quail-derived component substances in the sample to be detected in the step (1) according to a standard curve;
the reaction system of the ddPCR in the step (2) contains an upstream primer, a downstream primer and a probe for amplifying quail source, and the sequences of the upstream primer, the downstream primer and the probe are as follows:
and (3) probe: 5'-AGCAGCCAACACAGCACCCA-3', respectively;
an upstream primer: 5'-ACAGCAACGTTCTTCAGCTC-3', respectively;
a downstream primer: 5'-CTGGGAGCGCTTGCTTTATT-3', respectively;
the 5 'end of the probe is modified by FAM, and the 3' end is modified by BHQ 1.
2. The method according to claim 1, wherein the standard curve in step (3) is prepared by: pretreating a quail meat sample, weighing the mass of the sample, extracting DNA in the sample, determining the copy number of the DNA by ddPCR, and establishing a linear relation between the substance content of the quail-derived component and the copy number of the DNA to obtain a standard curve.
3. The method according to claim 2, wherein the standard curve is Gn = 2.85C + 37.915, where Gn is the copy number of the DNA of the sample and C is the quail-derived ingredient substance content.
4. The method according to claim 1 or 2, wherein the pretreatment is that the quail meat sample or the sample to be tested is divided into small pieces, and the small pieces are ground into powder after vacuum freeze drying.
5. The method according to claim 4, wherein the vacuum freeze drying is carried out under the conditions of a vacuum degree of 0.090-0.100 mbar, a temperature of-60-55 ℃ and a drying time of 48-72 h.
6. The method as claimed in claim 1 or 2, wherein the ddPCR reaction system has a total volume of 20 μ L, and comprises: ddPCR Super mix for Probes 10. mu.L, probe 0.5. mu.L, forward primer and reverse primer 1.8. mu.L each, DNA 1. mu.L, ddH2Make up to 20. mu.L of O.
7. The method of claim 6, wherein the initial concentration of the forward primer, the reverse primer and the probe is 10 μmol/L.
8. The method of claim 6, wherein the ddPCR amplification procedure is: 10 min at 95 ℃, 30 s at 94 ℃, 1 min at 60 ℃, 40 cycles, and 10 min at 98 ℃.
9. A quail-derived detection kit, which is characterized by comprising the upstream primer, the downstream primer and the probe of claim 1.
10. The detection kit according to claim 9, wherein the reaction system of the detection kit is as follows: ddPCR Super mix for Probes 10. mu.L, 0.5. mu.L of the probe of claim 1, 1.8. mu.L of each of the forward primer and the reverse primer of claim 1, 1. mu.L of the template DNA, ddH2Make up to 20. mu.L of O.
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