CN113278685A - Molecular identification method for storage life of pericarpium citri reticulatae - Google Patents
Molecular identification method for storage life of pericarpium citri reticulatae Download PDFInfo
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Abstract
The invention relates to the technical field of medicinal material detection, and discloses a molecular identification method for the storage life of pericarpium citri reticulatae, which comprises the following steps: (1) preparing a trnL-trnF fragment standard, and performing C of each diluted concentration standard by a fluorescent quantitative PCR methodqDetermination of the value, establishing the copy number logarithm of the trnL-trnF DNA fragment and the corresponding CqA standard curve of values; (2) extracting genome DNA of the citrus chachiensis peels with different storage years as a template, and determining C of trnL-trnFDNA fragment in the DNA template by a fluorescent quantitative PCR methodqValue, C obtained from samples of different storage yearsqSubstituting the value into the standard curve in the step (1) to calculate a logarithmic copy number value; (3) and performing linear regression analysis on the copy number logarithmic value and the storage life obtained by the trnL-trnF segments in the citrus tangerina of different storage lives, and establishing a variation curve of the citrus tangerina of different storage lives based on the DNA segment copy number judgment. The method can identify the storage life of the pericarpium citri reticulatae and provide basis and guarantee for high quality and high price of the pericarpium citri reticulatae.
Description
Technical Field
The invention relates to the technical field of medicinal material detection, in particular to a molecular identification method for the storage life of citrus grandis.
Background
The Citrus grandis is a famous medicinal material in China, is derived from dried mature pericarp of Citrus reticulata 'Chachi', is mainly produced in the New society of Guangdong, has the characteristics of medicine and food, and plays an important role in the market.
The tangerine peel is good for old people, namely, the tangerine peel can be stored for at least three years before being used as a medicine, and the price of the tangerine peel is higher as the storage year is longer. According to reports and surveys, the price of the euryale ferox peel stored for 3-5 years is generally 800 yuan/kg; about 1600 yuan/kg for 10 years, about 16000 yuan/kg for 20 years, 24000 yuan/kg for 25 years, and 68000 yuan/kg for 30 years. The pericarpium Citri Tangerinae of 50 years old or so is generally not less than 20 ten thousand yuan/kg. Therefore, the market often shows that the pericarp of other citrus is used as the citrus aurantium, the sun-curing year of the citrus aurantium is exaggerated, and the citrus aurantium with shorter storage year is used as the citrus aurantium for sale in high-age.
At present, the storage life of citrus chachiensis is mainly determined by discussing the relationship between the change rule of its chemical components and the storage life, such as FTIR, GC-MS and other analysis methods; in addition, researchers also use a spectrocolorimeter to research the correlation between the color characteristics and the storage life of citrus aurantium to judge the storage time of the long-storage traditional Chinese medicine. The research has reference significance for the year identification of the pericarpium citri reticulatae in the new conference, but the research samples are few, the specificity is poor, the uncontrollable property exists, and the practical application cannot be formed.
During storage of biological material, the DNA therein will gradually degrade, and the copy number of some DNA fragments therein will gradually decrease, with a regular quantity-time curve existing between the years of storage of the material. The storage environment of the citrus grandis is stable and the citrus grandis is subjected to a standard aging mode, so that the degradation curve is smooth, and the possibility of severe decline is low. In view of the above, the invention provides an objective method for clearly and reliably identifying the storage years of citrus grandis by using a molecular biology technology to determine the fragments with different copy numbers in citrus grandis with different storage years by using a fluorescent quantitative PCR method.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide an objective method for clearly and reliably identifying the storage years of citrus grandis by using a molecular biology technology. The copy number of the trnL-trnF segments in the citrus peel with different storage years is determined by adopting a fluorescent quantitative PCR method, the logarithmic value of the copy number has obvious correlation with the storage years, and the correlation coefficient is as high as 0.9748. The method can provide basis and guarantee for the high quality and the high price of the pericarpium citri reticulatae, and practically protect the rights and benefits of consumers; also can provide reference for determining the storage time of other Chinese medicinal materials.
The technical scheme of the invention is as follows:
the invention provides a molecular identification method for the storage life of citrus grandis, which comprises the following steps:
(1) preparing a trnL-trnF fragment standard, and performing C of each diluted concentration standard by a fluorescent quantitative PCR methodqDetermination of the value, establishing the copy number logarithm of the trnL-trnF DNA fragment and the corresponding CqA standard curve of values;
(2) extracting genome DNA of the citrus chachiensis peels with different storage years as a template, and determining C of trnL-trnFDNA fragment in the DNA template by a fluorescent quantitative PCR methodqValue, C obtained from samples of different storage yearsqSubstituting the value into the standard curve in the step (1) to calculate a logarithmic copy number value;
(3) and performing linear regression analysis on the copy number logarithmic value and the storage life obtained by the trnL-trnF segments in the citrus tangerina of different storage lives, and establishing a variation curve of the citrus tangerina of different storage lives based on the DNA segment copy number judgment.
In the technical scheme of the invention, the trnL-trnF fragment standard is based on an amplicon standard or a plasmid standard.
In the technical scheme of the invention, the amplification system of the fluorescent quantitative PCR reaction is as follows: DNA template 2. mu.L (containing 200ng of sample DNA); upstream and downstream primers were 0.6. mu.L each; takara TB Green Premix Ex Taq II 10. mu.L; deionized water 6.8. mu.L, total volume of reaction 20. mu.L.
In the technical scheme of the invention, the qPCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s, annealing at 59 ℃ for 1min for 40 cycles, and fluorescence signal was collected at the end of each cycle extension.
In the technical scheme of the invention, the citrus chachiensis peels with different storage years comprise citrus chachiensis peels with storage time of 1, 2, 3, 4, 5, 8 and 12 years.
In the technical scheme of the invention, the storage age is 1, 2, 3, 4, 5, 8 and 12 years and the storage age is subjected to linear regression analysis by taking the storage age as an abscissa, the lg copy number as an ordinate or the lg copy number as an abscissa and the storage age as an ordinate to finally generate a correlation curve.
In the technical scheme of the invention, no matter the standard curve is established based on the amplicon standard substance or the plasmid standard substance, the storage life and the copy number logarithmic value of the citrus grandis finally have good correlation.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention adopts a fluorescent quantitative PCR method to measure the copy number of trnL-trnF segments in the citrus peel with different storage life, the logarithm of the copy number has obvious correlation with the storage life, and the correlation coefficient is as high as 0.9748. The method can identify the storage life of the pericarpium citri reticulatae, provide basis and guarantee for high quality and high price of the pericarpium citri reticulatae, and practically protect consumers' rights and benefits; also can provide reference for determining the storage time of other Chinese medicinal materials.
2. The method established by the molecular identification method for the storage life of the citrus grandis has universal applicability, and the idea of judging the storage life of the citrus grandis by measuring the copy number is novel, so that the method becomes a new identification method or a supplement of the existing identification method for the storage life of the citrus grandis, and further provides basis and guarantee for the high-quality and high-price of the citrus grandis, and the rights and interests of consumers are practically protected.
Drawings
FIG. 1 shows the amplification curves for each DNA fragment standard (amplicon), wherein the A corresponds to trnL-trnF, the B corresponds to trnL, the C corresponds to ITS2, the D corresponds to psbA-trnH, the E corresponds to matK, and the F corresponds to rbcL.
FIG. 2 shows the melting curves of the respective fragment standards (amplicons), wherein A corresponds to the fragment trnL-trnF, B corresponds to the fragment trnL, C corresponds to the fragment ITS2, D corresponds to the fragment psbA-trnH, E corresponds to the fragment matK, and F corresponds to the fragment rbcL.
FIG. 3 is a graph of the correlation of the log copy number of each fragment in Potentilla anserina with the storage life (based on amplicon standards).
FIG. 4 is a plot of the correlation between the number of copies of the trnL-trnF fragment versus the storage life of Citrus aurantium (based on amplicon standards).
FIG. 5 is a plot of the log copy number of the trnL-trnF fragment of Citrus aurantium Linn vs. storage life (based on plasmid standards).
Detailed Description
In order to make the technical solutions of the present invention better understood and enable those skilled in the art to practice the present invention, the following embodiments are further described, but the present invention is not limited to the following embodiments.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. Unless otherwise specifically stated, the various starting materials, reagents, instruments and equipment used in the following examples of the present invention are either commercially available or prepared by conventional methods.
First, the following specifically exemplifies the technical scheme of the present invention with reference to specific embodiments:
example 1:
the material is pericarpium citri reticulatae which is stored in a new meeting area of Guangdong Jiangmen city for 1, 2, 3, 4, 5, 8 and 12 years. Wherein, the pericarpium citri reticulatae stored for 3 years has 2 batches of samples, and the pericarpium citri reticulatae stored for 2 years has 3 batches of samples. Primer information for the matK, rbcL, psbA-trnH, ITS2, trnL-trnF fragments is shown in Table 1 below:
TABLE 1 primer information Table
Example 2 extraction method of DNA:
the method comprises the following specific steps: weighing 50mg of pericarpium citri reticulatae sample, adding 20mg of PVP and a proper amount of liquid nitrogen, quickly grinding into powder, and transferring to a 2mL centrifuge tube; adding 1mL of precooled CTAB-free buffer solution (0.1mmol/L of 1.0mol/L Tris-HCl with the pH value of 8, 5mmol/L EDTA-2Na, 0.25mmol/L NaCl, 2% PVP and 0.4% beta-mercaptoethanol) into a centrifuge tube, shaking and uniformly mixing, centrifuging at 12000rpm for 5min, discarding the supernatant, and repeatedly washing for 2 times; adding 0.8mL of preheated 2% CTAB buffer solution (0.1mmol/L of 1.0mol/L Tris-HCl with pH 8, 20mmol/L EDTA-2Na, 1.4mmol/L NaCl, 2% PVP, 2% CTAB, 2% beta-mercaptoethanol), mixing, and then carrying out water bath at 65 ℃ for 60min, wherein the water bath is shaken for 1 time every 10 min; taking out the centrifuge tube, cooling to room temperature, adding chloroform-isoamylol (24: 1) with the same volume, reversing and mixing uniformly for 5min, centrifuging at 12000rpm for 5min, sucking the supernatant, placing the supernatant into a new 2mL centrifuge tube, repeating the steps once, sucking the supernatant, placing the supernatant into a new 7mL centrifuge tube, adding 3mol/L NaAc with one tenth of the volume and precooled absolute ethyl alcohol with twice of the volume, gently mixing uniformly until flocculent precipitates, centrifuging at 12000rpm for 5min, and discarding the supernatant; adding 0.8mL of precooled 75% ethanol, obliquely placing and standing for 5min, centrifuging at 12000rpm for 2min, discarding the supernatant, and repeating for 1 time; ethanol was air-dried at room temperature, 100. mu.L of TE was added, and the mixture was dissolved in a refrigerator at 4 ℃ and used.
Example 3 construction of amplicon standards:
amplicons of each fragment were prepared. The PCR volume was 15. mu.L, and the system contained 2 XTaq Plus PCR MasterMix 6.5. mu.L, upstream and downstream primers 0.5. mu.L each (final concentration 250nmol/L), deionized water 6.5. mu.L, and DNA template 1. mu.L. The PCR amplification procedure was the procedure for each primer (Table 2). And purifying the amplified product by using a DNA purification recovery kit, sequencing and identifying the amplified product to obtain a required fragment, using the fragment as a standard substance, and measuring the DNA concentration of the standard substance by using a NanoDrop 2000 ultramicro ultraviolet spectrophotometer. Using the formula N (copies/. mu.L) ═ 6.02X 1023)×(ng/μL×10-9) /(DNA length. times.660) the copy number was calculated.
TABLE 2 PCR amplification conditions
Example 4 creation of standard curve:
establishment of a standard curve: the established standards were diluted in 10-fold gradients for qPCR assays. The total qPCR reaction volume was 20 μ L: 2 μ L of template, 0.6 μ L of each of upstream and downstream primers, 10 μ L of Takara SYBR Green Premix Ex Taq II, and 6.8 μ L of deionized water. The experiment is in Roche-
And (3) performing on a 96 real-time fluorescent quantitative PCR instrument. The qPCR amplification program was set up differently for each primer (table 3) and fluorescence signals were collected at the end of each cycle extension. The melting curve program was: 1s at 95 ℃; 15s at 65 ℃ and 1s at 95 ℃. In the experiment, 3 duplicate wells were made for each sample in the same experiment, and a blank control group without any species of DNA template (ultrapure water) added was set. Finally, the logarithmic value of the copy number of the standard product (base 10, i.e. lg copy number) is compared with the corresponding CqLinear regression analysis of the values, lg copy number as abscissa, CqThe values are the ordinate and the standard curve is finally generated. In the experiment, 3 duplicate wells were made for each sample in the same experiment, and a blank control group without any species DNA template (double distilled water substitution) was set.
TABLE 3 qPCR amplification conditions
Example 5 determination of copy number of samples for different storage life:
samples of citrus grandis with different storage lives are taken, 3 samples of each sample are taken in parallel, DNA is extracted as a template (the amount of the added DNA is 200ng) according to the embodiment 2 to perform the measurement of fluorescence quantitative PCR, and the logarithmic value (with 10 as the base, namely the lg copy number) of the corresponding storage life and the copy number of each sample is subjected to linear regression analysis. And carrying out subsequent methodology investigation on the DNA fragment with good correlation and better amplification specificity.
Example 6 stability, reproducibility and recovery:
taking DNA of pericarpium Citri Tangerinae sample with storage life of 4 years, performing qPCR independent repeat test for 6 times at different time periods of 0, 2, 4, 8, 12, and 24h, and calculating test time CqValue RSD, inter-group variance within the group was analyzed to verify the stability of the method. In order to test the repeatability, 6 parts of pericarpium citri reticulatae samples with the storage life of 4 years are taken, DNA is extracted according to the embodiment 2, then the fluorescence quantitative PCR reaction is carried out, 3 multiple holes are made for each sample in the same experiment, and the RSD value of 6 batches of samples is calculated. The theoretical concentration is 1.78 multiplied by 106C detected by directly carrying out fluorescence quantitative PCR on copies/mu L standard substanceqSetting the value average value as theoretical value, adding isovolumetric standard substance into blank matrix extract, performing fluorescent quantitative PCR amplification by blank standard-adding recovery method to obtain CqValues are measured values and the recovery (measured value/theoretical value x 100%) is calculated.
Example 7 construction based on plasmid standards:
the DNA fragment (trnL-trnF fragment) having the above-mentioned correlation and amplification specificity was ligated to pET-28a (+) vector to obtain a plasmid standard containing the DNA fragment, and then the experiments of example 4 and example 5 were carried out.
Second, results and analysis
1. Amplification curve and melting curve
The typical S-type fluorescence signal curve is amplified by the amplicon standard of each primer, the intervals of the dilution gradients are equal, the multi-well amplification of each standard is normal, and the fitting degree is high (figure 1). The melting curves for psbA-trnH, matK, and rbcL show two melting peaks, indicating that there is non-specific amplification in their qPCR amplification reactions. the melting curves for the trnL-trnF, trnL and ITS2 fragment standards were all unimodal (FIG. 2). However, the trnL fragment negative control also has a fluorescence signal, and the melting peak of the negative control and the melting peak of the sample appear at the same position, so that a single melting peak appears in the figure.
2. Linearity and sensitivity
The concentration of the purified sample was 5.1-18.1ng/uL as determined by a NanoDrop 2000 UV spectrophotometer, with a logarithmic transformation range of 6.88-11.22 per initial copy number. Each one ofThe standard curve established by the segments shows good linear relation, R2Are all greater than 0.99 (table 4). the linear range of trnL-trnF is 109-103The linear ranges of copies/μ L, matK, rbcL, ITS2, psbA-trnL and trnL are respectively 10-2-105、105-1010、103-107、104-109、105-1010copies/μL。
TABLE 4 Standard curves for the respective fragment standards
3 establishing the correlation curve of the copy number and the storage life of the pericarpium citri reticulatae with different storage lives
The results show that, in addition to trnL-trnF and trnL, the log copy number of the other four fragments correlates poorly with storage time (FIG. 3). the copy number logarithm values of trnL-trnF and trnL are in a tendency of gradually decreasing in the storage process, the correlation coefficient between the copy number logarithm value of trnL fragments and the storage life is 0.824, but non-specific amplification products (melting peaks appear in negative control) exist during the amplification of the trnL fragments, and the sample measurement value can be influenced, so that the consideration is not carried out. the trnL-trnF fragment was selected for subsequent stability, reproducibility and recovery tests (FIG. 4) because of its good specificity when amplified (single melting peak) and a correlation coefficient between log copy number and storage life of 0.9748 (linear equation of y ═ 0.312x + 7.4278). In addition, log copy number values of the pericarpium citri reticulatae stored for 1-12 years obtained on the basis of a standard curve established by a trnL-trnF fragment plasmid standard substance also obtain good correlation with the storage life, and the correlation is 0.967 (figure 5).
4. Stability, reproducibility and recovery
And (4) performing methodology investigation on a correlation curve of the copy number logarithm of the citrus tangerina with different storage life spans and the storage life span established based on the trnL-trnF segment. In stability examination, the RSD value in the same sample group is 0.05-0.45%, the RSD in the groups is 1.10%, and both are less than 3%, which indicates that the formula is adoptedThe process had good stability over 24h (Table 5). The RSD values of 6 replicates ranged from 0.06 to 1.09% (Table 6). According to the requirement of the recovery rate limit of 2020 edition of Chinese pharmacopoeia, the content of the standard substance is lower than 0.0001% (here, the content of the standard substance in each 100 ml of solution is lower than 10)-9g) The recovery rate of the sample is required to be 70-125%, and the theoretical concentration of the invention is 8.51 multiplied by 105The average recovery rate of copies/muL standard DNA is 75.43% (Table 7), which meets the requirement of recovery rate limit, and shows that the detection method has good accuracy.
TABLE 5 stability test of Guangzhou orange peel with 4 years of storage (n ═ 3)
TABLE 6 repeatability test of Guangzhou orange peel with 4 years of storage (n ═ 3)
TABLE 7 fluorescent quantitative PCR sample-adding recovery test
From the above analysis, it is known that the log values of the copy numbers of four DNA fragments, i.e., matK, rbcL, psbA-trnH and ITS2, in Citrus reticulata Blanco with different storage life are less correlated with the storage life, while the log values of the copy numbers of the trnL fragment are better correlated with the storage life (R20.8529) but there is non-specific amplification. Therefore, five DNA fragments of matK, rbcL, psbA-trnH, ITS2 and trnL are not suitable for identifying the storage life of the euryphylla. The log value of the copy number of the trnL-trnF segment in the citrus reticulata blanco with different storage years has good correlation with the storage years (R)20.9748) and the amplification specificity, a new molecular identification method for judging the storage life of the citrus grandis based on the copy number logarithmic value of the trnL-trnF fragment can be established.
In conclusion, the established method is accurate, stable, good in reproducibility and universal in applicability, the idea of judging the storage life of the pericarpium citri reticulatae by measuring the copy number is novel, and the method can be used as a new identification method or a supplement of the existing method for identifying the storage life of the pericarpium citri reticulatae, so that the basis and guarantee are further provided for the high-quality and high-price of the pericarpium citri reticulatae, and the rights and interests of consumers are practically protected.
It should be noted that when ranges are recited herein, unless otherwise stated, each endpoint, and any value between the endpoints, of each range can be selected. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (6)
1. The molecular identification method for the storage life of pericarpium citri reticulatae is characterized by comprising the following steps of:
(1) preparing trnL-trnF fragment standard substance, and performing C of each diluted concentration standard substance by a fluorescent quantitative PCR methodqDetermination of the value, establishing the copy number logarithm of the trnL-trnF DNA fragment and the corresponding CqA standard curve of values;
(2) extracting genome DNA of the citrus chachiensis peels with different storage years as a template, and determining C of trnL-trnF segment in the DNA template by a fluorescent quantitative PCR methodqValue, C obtained from samples of different storage yearsqSubstituting values into step (1)Calculating a logarithmic copy number value by using a standard curve;
(3) and performing linear regression analysis on the copy number logarithmic value and the storage life obtained by the trnL-trnF segments in the citrus tangerina of different storage lives, and establishing a correlation curve for judging the citrus tangerina of different storage lives based on the copy number of the DNA segment.
2. The method of claim 1, wherein the trnL-trnF fragment standard is an amplicon-based standard or a plasmid-based standard.
3. The method for identifying the storage life of citrus grandis according to claim 1, wherein the amplification system of the fluorescent quantitative PCR reaction is: DNA template 2. mu.L (containing 200ng of sample DNA); upstream and downstream primers were 0.6. mu.L each; takara TB Green Premix Ex Taq II 10. mu.L; deionized water 6.8. mu.L, total volume of reaction 20. mu.L.
4. The method for identifying the storage life of citrus grandis according to claim 1, wherein the qPCR amplification procedure comprises: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s, annealing at 59 ℃ for 1min for 40 cycles, and fluorescence signal was collected at the end of each cycle extension.
5. The method of claim 1, wherein the citrus chachiensis peels stored for different storage periods comprise citrus chachiensis peels stored for 1, 2, 3, 4, 5, 8 and 12 years.
6. The method of claim 1, wherein the log of the storage life of pericarpium Citri Reticulatae Chachiensis is 1, 2, 3, 4, 5, 8, 12 years old and the storage life is linear regression analysis using the storage life as abscissa, lg copy number as ordinate or lg copy number as abscissa and the storage life as ordinate to generate the correlation curve.
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| CN114814003A (en) * | 2022-04-01 | 2022-07-29 | 广州白云山陈李济药厂有限公司 | Quantitative detection method for aging degree of dried orange peel |
| CN117187436A (en) * | 2023-09-25 | 2023-12-08 | 广州白云山陈李济药厂有限公司 | Method for detecting ageing years of pericarpium citri reticulatae |
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