CN109943625B - Real-time fluorescence PCR detection method for donkey-derived ingredients in food and feed - Google Patents
Real-time fluorescence PCR detection method for donkey-derived ingredients in food and feed Download PDFInfo
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Abstract
The invention discloses a real-time fluorescence PCR detection method of donkey-derived components in food and feed, which comprises the following steps: the first step, taking DNA of a sample to be detected as a template, and carrying out fluorescent quantitative PCR amplification to obtain a PCR amplification product; secondly, detecting the fluorescence signal of the amplification product; thirdly, judging whether the sample contains donkey-derived components or not according to the Ct value of the detection result and quantitatively detecting the content of the donkey-derived components in the sample; wherein, the reaction system for PCR amplification contains a specific primer pair for amplifying donkey-derived components and a specific probe for donkey-derived components. The specific primer pair and the probe for real-time fluorescent PCR amplification of donkey-derived components have good specificity and high sensitivity, provide a quantitative detection method for rapidly and accurately detecting whether the donkey-derived components are contained in food and feed, and have good application prospect.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a real-time fluorescence PCR detection method for donkey-derived components in food and feed.
Background
The identification and quantification of the type of meat products plays an important role in food safety monitoring, and in some european countries, all meat products need to be explicitly labeled with animal-derived ingredients and their proportions, even if the meat is adulterated, it still happens. Due to the factors of similar meat textures and the like, consumers cannot accurately identify the meat types, illegal vendors have a good opportunity, and the mishap of the meat does not influence health, but the distrust of the consumers is greatly increased. Therefore, it is necessary to establish a set of accurate and reliable qualitative detection method, and it is not easy to establish.
At present, primers and probes for detecting animal components at home and abroad are generally designed aiming at mitochondrial genes, and the copy number of the mitochondrial genes in cells is 5000-6000 copies or more. In the actual research work, it is found that when the primer and the probe are designed by using multi-copy mitochondrial genes for detection, a lower Ct value can also appear under the condition of extremely low concentration of a target detection object, so that a detection person can hardly judge whether an actual sample really contains a target component or the sample is caused by very slight pollution. Therefore, when the primer and the probe are designed by utilizing the mitochondrial gene for detection, false positive results are easy to appear or the detection results with low target content are difficult to judge, and the like, and the error of the detection results can cause great trade disputes.
Therefore, a set of real-time fluorescence PCR detection method for donkey-derived components in food and feed, which has good specificity and high sensitivity and can avoid false positive results, is necessary to be established.
Disclosure of Invention
The invention aims to provide a real-time fluorescence PCR detection method for donkey-derived ingredients in food and feed, so as to overcome the defects that false positive results are easy to occur in the prior art and the like. The second purpose of the invention is to provide a detection kit and application of the detection kit. The third purpose of the invention is to provide a primer pair and a probe of the real-time fluorescence PCR detection method for donkey-derived components in food and feed.
In order to achieve the purpose, the invention adopts the following technical scheme:
as a first aspect of the invention, a real-time fluorescence PCR detection method for donkey-derived ingredients in food and feed comprises the following steps:
the first step, taking DNA of a sample to be detected as a template, and carrying out fluorescent quantitative PCR amplification to obtain a PCR amplification product;
secondly, detecting the fluorescence signal of the amplification product;
thirdly, judging whether the sample contains donkey-derived components or not according to the Ct value of the detection result and quantitatively detecting the content of the donkey-derived components in the sample;
the reaction system for PCR amplification contains a specific primer pair for amplifying donkey-derived components and a specific probe for donkey-derived components, the sequence of the specific primer pair for amplifying donkey-derived components is shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the specific probe for amplifying donkey-derived components is shown as SEQ ID NO. 3.
According to the invention, the conditions for the PCR amplification are as follows:
the reaction system is 25 μ L: PCR enzyme mixture 12.5. mu.L, primers 10. mu.M 1. mu.L each, probe 10. mu.M 0.5. mu.L, DNA template 5. mu.L, PCR grade water 5. mu.L;
and (3) PCR reaction conditions: 10min at 95 ℃; 15s at 95 ℃ and 60s at 60 ℃; for a total of 45 cycles.
As a second aspect of the present invention, a detection kit for donkey-derived ingredients, comprising the following reagents:
(a) amplifying a specific primer pair of donkey-derived components;
(b) a specific probe of donkey-derived components;
wherein, the sequence of the specific primer pair for amplifying the donkey-derived component is shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the specific probe for amplifying the donkey-derived component is shown as SEQ ID NO. 3.
Further, the detection kit further comprises:
(c) and the standard reference substance is used for quantitatively detecting the content of the donkey-derived component in the sample.
As a third aspect of the invention, the application of the detection kit is provided, and the detection kit is used for qualitatively detecting donkey-derived ingredients in food or feed.
As a fourth aspect of the invention, a specific primer pair and a probe of donkey-derived components are provided, wherein the sequence of the specific primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the specific probe is shown as SEQ ID NO. 3.
The invention has the beneficial effects that:
1. the specificity is good, specific amplification can be realized for donkey-derived components, and specific amplification cannot be realized for other components except donkey; moreover, the primer pair and the probe have good stability and intraspecies conservation.
2. The primer pair and the probe can be used for rapidly detecting whether donkey-derived components exist in food or feed, and the primer pair and the probe are simple to operate and high in sensitivity.
3. The real-time fluorescence PCR detection method of donkey-derived ingredients has wide application range, is particularly suitable for detecting donkey-derived ingredients in various foods or feeds, can be popularized and applied, provides technical support for guaranteeing the quality of products, protecting the right of knowledge and selection of consumers, maintaining normal economic order and the like, and provides technical support for food market supervision departments and inspection and quarantine departments.
Drawings
FIG. 1 is a graph of real-time fluorescent PCR sensitivity assay amplification curves for the first set of primer pairs and probes of example 3. Wherein, the amplification curves are 40 ng/. mu.L, 20 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 0.1 ng/. mu.L and 0.01 ng/. mu.L donkey DNA samples from left to right respectively.
FIG. 2 is a graph of real-time fluorescent PCR sensitivity assay amplification curves for the second set of primer pairs and probes of example 3. Wherein, the amplification curves are 40 ng/. mu.L, 20 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 0.1 ng/. mu.L and 0.01 ng/. mu.L donkey DNA samples from left to right respectively.
FIG. 3 is a graph of amplification curves for the specificity assay of example 4.
FIG. 4 is a quantitative calibration curve of donkey-derived component of example 8.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The invention screens out the single copy nuclear gene with both interspecific specificity and intraspecies conservation as a target gene, utilizes the real-time fluorescence PCR detection technology to detect donkey-derived ingredients in food and feed, and comprehensively verifies the specificity, conservation, sensitivity and the like of the established method through a synergistic experiment.
The experimental materials of the following examples are as follows:
1. PCR enzyme mixtures were purchased from Heracles, Calif. (Bio-Rad, Hercules, Calif., USA);
2. PCR grade water was purchased from SAMMER Scientific, Salt Lake City, UT, USA, Utah;
3. TE was purchased from Tiangen Biotech (Beijing) Ltd (Tiangen Biotech, Beijing, China).
Example 1 design of primer pairs and probes
(1) Design of first set of primer probes
Donkey genome scaffold gene (GenBank: NW _014638576.1) was chosen as target gene design primer and probe. The sequence was first analyzed using NCBI and then the Primer from Applied Biosystems (ABI; Foster City, Calif., USA) was used
Software version 3.0 was designed, the amplified fragment length for donkey-derived component was 95bp (located in the interval of nucleotide 581564 and 581658), and the primer probe sequences are shown in Table 1.
TABLE 1 first set of primer Probe sequences
(2) Design of second set of primer probes
According to the primer probe design method, partial fragments in donkey genome scaffold gene (GenBank: NW _014638576.1) are selected to design primer pairs and probes, the amplification fragment length of donkey-derived components is 108bp, and the sequences of the primer probes are shown in Table 2.
TABLE 2 second set of primer Probe sequences
(3) The total volume of the reaction was 25. mu.l, the reaction system is shown in Table 3, and the reaction procedure is shown in Table 4.
TABLE 3 PCR reaction System
TABLE 4 PCR reaction procedure
Example 2 sample preparation, DNA extraction and real-time fluorescent PCR assay
(1) Preparation of samples
Fresh lean meat and other commercial meat products are purchased from slaughterhouses and supermarkets. Respectively cut, dried in an oven (UFE500 AO; memoert, germany) at 80 ℃ for 72 hours, and then pulverized into ultrafine powder using a liquid nitrogen pulverizer (SPEX SamplePrep, usa).
(2) Extraction of DNA
DNA extraction method using phenol chloroform 100mg of sample was extracted: adding 800 μ l of rhizoma corydalis lysate and 10 μ l of proteinase K, vortex shaking, mixing, soaking in 65 deg.C water for 60min, adding equal volume of phenol and chloroform, mixing, centrifuging at 12000rpm for 10min, collecting supernatant, purifying with phenol and chloroform, and adding one tenth volume of 3M sodium acetate (pH 5.2). Followed by precipitation with two volumes of ice-bath absolute ethanol at-20 ℃ for 30min, followed by high speed centrifugation at 12000rpm at 4 ℃ for 30 min. After rinsing twice with 75% ethanol, it was dried at room temperature, and finally 100. mu.l of ultrapure water was added and stored.
(3) Real-time fluorescent PCR assay
Prior to use, the reagents are thawed and centrifuged for use. Ensure that each reagent was well mixed prior to pipetting. The PCR reaction mixture should contain all the components necessary for the PCR reaction, except for the sample DNA. The amount of PCR reaction mixture required depends on the number of samples, and at least the amount of mixture required to be more than the number of samples is used to eliminate pipetting losses. After the premix was mixed well, 20. mu.l of the mixture was transferred to each reaction tube, and 5. mu.l of DNA was added as a template. The positive control, negative control, extraction blank and blank were used, plus 5. mu.l each. It should be noted that a PCR inhibition control can also be established. Starting the instrument to perform real-time fluorescent quantitative PCR reaction.
Example 3 sensitive detection of primer pairs and probes for donkey-derived component
Donkey genomic DNA was extracted by the DNA extraction method of example 2, diluted to 40 ng/. mu.l, 20 ng/. mu.l, 10 ng/. mu.l, 1 ng/. mu.l, 0.1 ng/. mu.l, 0.01 ng/. mu.l, 0.001 ng/. mu.l and 0.0001 ng/. mu.l, and then subjected to real-time fluorescence PCR assay using the first and second sets of primer probes of example 1, respectively, and the assay was repeated 6 times. The results are shown in FIGS. 1 and 2.
FIG. 1 shows that in 6 tests, amplification curves appeared in 40 ng/. mu.l, 20 ng/. mu.l, 10 ng/. mu.l, 1 ng/. mu.l, 0.1 ng/. mu.l and 0.01 ng/. mu.l donkey sample DNA, while no amplification curves appeared in 0.001 ng/. mu.l and 0.0001 ng/. mu.l donkey sample DNA.
FIG. 2 shows that in 6 assays amplification curves appeared in 40 ng/. mu.l, 20 ng/. mu.l, 10 ng/. mu.l, 1 ng/. mu.l, 0.1 ng/. mu.l and 0.01 ng/. mu.l of sample DNA, but the amplification curves were not proportional to DNA concentration.
And (4) conclusion: 1. the real-time fluorescence PCR detection of donkey-derived components is carried out by adopting a first group of primer probes, an obvious S-shaped amplification curve is obtained when the usage amount of a template is 0.01 ng/mu l, and the detection sensitivity reaches 0.01 ng/mu l;
2. the real-time fluorescence PCR detection of donkey-derived components is carried out by adopting a second group of primer probes, and the detection effect is unstable;
3. the first group of primer probes and the second group of primer probes are obviously different, and compared with the second group of primer probes, the first group of primer probes has better accuracy when being used for real-time fluorescent PCR detection of donkey-derived components. Subsequent tests were therefore performed only for the first set of primer pairs and probes.
Example 4 specific detection of primer pairs and probes for donkey-derived Components
(1) This example uses primer pairs and probes of donkey-derived ingredients of example 1 to detect 41 species of DNA samples (including genome DNA of donkey of 5 strains and genome DNA of 36 other animal and plant species (20 ng/. mu.l)) of Table 4, and uses the extraction method of example 2 to extract DNA and uses real-time fluorescence PCR detection technology to verify the inter-species specificity of the designed primers and probes. The results are shown in Table 5 and FIG. 3.
(2) The intraspecific conservation of the primer and probe sets of the donkey is verified by selecting 5 strains of donkey (Guanzhong donkey, Qingyang donkey, Xinjiang donkey, southwest donkey and Jinnan donkey), and the result shows that the 5 strains are detected. The results are shown in Table 5 and FIG. 3.
TABLE 5 detection results of inter-species specificity and intra-species conservation of primers and probes
And (4) conclusion: (1) the primer and the probe do not generate cross reaction with other species, and have good species specificity.
(2) The verification results of Guanzhong donkey, Qingyang donkey, Xinjiang donkey, southwest donkey and Jinnan donkey show that the designed primers and probes have good intraspecies conservation.
Example 5 stability testing of primer pairs and probes
The stability of the method was verified by the following changes in the influencing factors: real-time fluorescent PCR instruments (ABI 7500, BioRad CFX96, ABI 7900HT Fast, Eppendorf Realplex 4); reaction volume (19. mu.l or 21. mu.l enzyme mix + 5. mu.l DNA sample); annealing temperatures (59 ℃ and 61 ℃); primer or probe concentrations (30% reduction, respectively). 3 PCR repeated analyses are carried out on each influencing factor, donkey genome DNA is used as a sample for verification, and the DNA content is 20 ng/. mu.l. The results are shown in Table 6.
TABLE 6 stability verification results for primers and probes
The results show that: changes of influencing factors such as instrument and equipment, reaction system volume, annealing temperature, primer probe concentration and the like do not obviously influence the method, and all donkey-derived components show positive results.
And (4) conclusion: the method of the invention has good stability.
Example 6 false positive and false negative validation of primer pairs and probes
The present example organises validation work on international cooperative experiments involving 12 laboratories. Each participating unit received 12 DNA samples for false positive and false negative verification experiments. All samples were randomly numbered, including 6 positive samples and 6 negative samples. Wherein, the concentration of the target gene of the 6 donkey-derived DNA solutions is 10 copies/mu l, and the concentration of the target gene of the 6 bovine-derived DNA solutions is 20 copies/mu l, and the PCR enzyme mixture, the primers and the probe sets are sent together with the sample.
Donkey-derived and bovine-derived DNA samples were extracted from donkey meat and beef, respectively (using the method of example 2), dissolved in 0.2X TE, and quantified to 10 copies/. mu.l and 20 copies/. mu.l by digital PCR. A false positive and false negative verification experiment was performed according to the reaction system and reaction conditions of example 1. The results are shown in Table 7.
TABLE 7 false positive and false negative cooperative experiment verification results
The results show that: all positive samples were tested positive and all negative samples were tested negative.
And (4) conclusion: the detection method has good accuracy.
Example 7 detection Limit (LOD) and Positive detectable Rate (POD) detection of primer pairs and probes
Verification of sensitivity experiments used plasmids as DNA templates into which the target gene fragments had been cloned, diluted with 20 ng/. mu.l salmon sperm DNA dissolved in 0.2X TE solution, and quantified plasmid DNA concentrations at 1000 copies/. mu.l as initial concentrations using digital PCR. The participating units were self-diluted with 20 ng/. mu.l salmon sperm DNA in a gradient to 7 concentrations of 4, 2, 1, 0.4, 0.2, 0.1 and 0.02 copies/. mu.l, each concentration being 6 PCR replicates.
(1) The statistics of the results of the international cooperative experiments show that the detection results are shown in Table 8.
TABLE 8 synergistic test verification results for lower limit of detection LOD 95%
| Donkey genome scaffold gene copy number | Positive result (C)t<45) |
| 20 | 72 |
| 10 | 72 |
| 5 | 72 |
| 2 | 67 |
| 1 | 54 |
| 0.5 | 27 |
| 0.1 | 4 |
The results show that: absolute LOD of the method95％≤5DNA copies。
And (4) conclusion: the detection method has higher sensitivity.
(2) The positive detection probability POD of the method is verified by using a new statistical model. The results are shown in Table 9.
TABLE 9 Positive detection probability POD results of the synergistic experiment
The results show that: the standard deviation of the method between laboratories at POD of 0.95 is 0.30 by the verification of cooperative experiments.
Example 8 quantitative detection of primer pairs and probes for donkey-derived component
Referring to the DNA extraction method of example 2, donkey genomic DNA was extracted, diluted to 100 ng/. mu.l, 10 ng/. mu.l, 1 ng/. mu.l, 0.1 ng/. mu.l, 0.01 ng/. mu.l for five concentration gradients, and detected using the primer pairs and probes of Table 1 at 6 replicates per concentration to obtain Ct values. The negative control was water. Thus, a quantitative standard curve is made and verified.
(1) Quantitative detection of donkey-derived component primer pair and probe
The results are shown in Table 10. Where the Ct value is the average of 6 replicates.
TABLE 10 Ct values for real-time fluorescent PCR quantitative determination assays
| Template dose (ng/ul) | Ct value |
| 100 | 23.515 |
| 10 | 27.276 |
| 1 | 30.716 |
| 0.1 | 33.598 |
| 0.01 | 37.867 |
| Negative control | - |
And (4) conclusion: it can be seen that the Ct value of amplification is increased in a gradient way and presents a certain linear relation along with the decrease of the concentration of donkey-derived ingredientsIs (R)2＝0.997)。
(2) Processing and computing of data
The results are shown in FIG. 4, where the logarithm of the DNA concentration is plotted as the abscissa and the average of the Ct values of 6 replicates of the sample is plotted as the ordinate, and curve fitting is performed on the points to obtain a quantitative standard straight line, the linear equation is that y is 3.5026x +20.087, and R is20.997. For the sample needing to be quantified, the respective Ct value of the sample to be measured is measured each time, and the corresponding x value is obtained by utilizing a standard curve formula. The donkey-derived component content of the sample to be detected can be obtained.
(3) Verification of quantitative standard curves
Diluting the extracted donkey genome DNA to two concentrations of 50 ng/mu l and 0.5 ng/mu l, repeating each concentration for 6 times, detecting respective Ct value, and calculating the donkey-derived component content by a quantitative standard curve to verify the repeatability of the method.
The results showed that the average of the values measured for the 50ng sample was 51.33ng and the average of the values measured for the 0.5ng sample was 0.47 ng.
And (4) conclusion: the method of the present example is demonstrated to have the ability to quantify accurately over a dynamic range. Therefore, the actually measured value of the method is consistent with the theoretical value, and the repeatability is good.
Example 9 actual product validation of primer pairs and probes for donkey-derived ingredients
Samples of 19 meat based food and animal feed samples collected in the market, import and export trade routes were tested using the first set of primers and probes of example 1 and the method of example 2. The results are shown in Table 11.
Table 1119 test results of donkey-derived ingredients of samples to be tested
| Serial number | Sample name | Test results |
| 1 | Sauce-flavor donkey meat | + |
| 2 | Donkey meat baked with hot fire | + |
| 3 | Beef-flavored cat food | — |
| 4 | Beef ham sausage | — |
| 5 | Beef luncheon meat | — |
| 6 | Braised beef can | — |
| 7 | Dried beef | — |
| 8 | Curry beef sauce | — |
| 9 | Beef-flavored dog food | — |
| 10 | Mutton-flavored dog food | — |
| 11 | Mutton chop | — |
| 12 | Chicken ham sausage | — |
| 13 | Pork ham sausage | — |
| 14 | Pork luncheon meat | — |
| 15 | Dried pork slice | — |
| 16 | Beef ball | — |
| 17 | Chicken-flavored dog food | — |
| 18 | Canned food "guan fish cat | — |
| 19 | Australian pet-grade chicken bone meal | — |
The results show that donkey-derived components are detected in the sauce-flavor donkey meat and donkey meat fire-cooking; no donkey-derived component was detected in the remaining food and feed.
And (4) conclusion: the meat quality source of the sample is consistent with the detection result. Meanwhile, the detection method of the embodiment 2 is also used for detecting whether donkey-derived ingredients exist in food and feed in large batch.
In conclusion, the detection method disclosed by the invention selects the single-copy nuclear gene as a target gene and utilizes a real-time fluorescence PCR technology to detect donkey-derived components in food and feed, and the specificity, stability, false positive and false negative, lower limit of detection (LOD) and positive detectable rate (POD) of the method are respectively verified by developing an international cooperative experiment, so that the result shows that the method has good interspecific specificity and interspecific conservation, higher stability, accuracy and sensitivity, and the lower limit of detection is 5 copies; the establishment of the detection method can also be applied to the detection of animal-derived components of other meat foods and feeds, and provides technical support for market supervision and related law enforcement of food and feed safety in China. Furthermore, the specific primer pair and the probe can be further prepared into a detection kit by a method known in the art, and the detection kit can further comprise a standard reference substance in addition to the specific primer pair and the probe, which is obvious to those skilled in the art.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
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Claims (6)
1. A real-time fluorescence PCR detection method for donkey-derived ingredients in food and feed is characterized by comprising the following steps:
the first step, taking DNA of a sample to be detected as a template, and carrying out fluorescent quantitative PCR amplification to obtain a PCR amplification product;
secondly, detecting the fluorescence signal of the amplification product;
thirdly, judging whether the sample contains donkey-derived components or not according to the Ct value of the detection result and quantitatively detecting the content of the donkey-derived components in the sample;
the reaction system for PCR amplification contains a specific primer pair for amplifying donkey-derived components and a specific probe for detecting donkey-derived components, the sequence of the specific primer pair for amplifying donkey-derived components is shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the specific probe for detecting donkey-derived components is shown as SEQ ID NO. 3.
2. The real-time fluorescent PCR assay of claim 1 wherein the PCR amplification conditions are as follows:
the reaction system is 25 μ L: PCR enzyme mixture 12.5. mu.L, primers 10. mu.M, each 1. mu.L, probes 10. mu.M, 0.5. mu.L, DNA template 5. mu.L, PCR grade water 5. mu.L;
and (3) PCR reaction conditions: 10min at 95 ℃; 15s at 95 ℃ and 60s at 60 ℃; for a total of 45 cycles.
3. A detection kit for donkey-derived components is characterized by comprising the following reagents:
(a) amplifying a specific primer pair of donkey-derived components;
(b) a specific probe for detecting donkey-derived components;
wherein, the sequence of the specific primer pair for amplifying the donkey-derived component is shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the specific probe for detecting the donkey-derived component is shown as SEQ ID NO. 3.
4. The test kit of claim 3, further comprising:
(c) a standard reference.
5. Use of the test kit according to claim 3 or 4 for qualitative or quantitative detection of donkey-derived ingredients in food or feed.
6. A specific primer pair and a probe for detecting donkey-derived ingredients are characterized in that the sequences of the specific primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the specific probe is shown as SEQ ID NO. 3.
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