CN114292921B - Method for quality control of Jinlong capsules by detecting copy number of Gekko Swinhonis specific fragment based on molecular quantification technology, primer and probe - Google Patents
Method for quality control of Jinlong capsules by detecting copy number of Gekko Swinhonis specific fragment based on molecular quantification technology, primer and probe Download PDFInfo
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Abstract
The invention discloses a method for quality control of gold dragon capsules by detecting the copy number of a gecko specific fragment based on a molecular quantification technology, and a primer and a probe. Comprising the following steps: designing and synthesizing specific primers and probes according to the mitochondrial genome sequence difference of the gecko original animal webless gecko and the mixed imitative product; extracting DNA of qualified products of the Jinlong capsules, and determining the copy number range of the specific fragments of the gecko without web in the qualified products of the Jinlong capsules through qPCR or ddPCR; extracting DNA of a gold dragon capsule sample to be detected, performing qPCR or ddPCR, and calculating the copy number of the specific fragment of the webless gecko; and (5) comparing with qualified products. The invention takes the copy number of the specific fragment of the webless gecko measured by qPCR or ddPCR as a quality control detection index, and examines the quality of the Jinlong capsule by comparing the copy number of the specific fragment in the Jinlong capsule to be detected with the copy number range of qualified products. The method can rapidly and sensitively specifically detect the Gekko Swinhonis component and content in the Jinlong capsule.
Description
Technical Field
The invention relates to the technical field of quality control of animal Chinese patent medicines, in particular to a method for controlling the quality of Jinlong capsules by detecting the copy number of a Gekko Swinhonis specific fragment based on a molecular quantification technology, and primers and probes.
Background
The Jinlong capsule consists of 2:1:1 of fresh gecko, fresh Bungarus Parvus and fresh Agkistrodon, is a cancer drug prepared from fresh medicinal animal formulas approved by the first example of China, has the effects of removing blood stasis and resolving hard mass, resolving depression and dredging collaterals, and is mainly used for primary liver cancer blood stasis and resolving hard mass, symptoms of right hypochondrium accumulation, chest hypochondrium pain, listlessness, abdominal distention, anorexia and the like. Gecko is a generic term for all lizards of the sub-order lizaridae (Sauria) Gekkonidae (Gekkonidae), also known as Gekkonidae, gekkonidae. Modern researches have found that gecko has anti-tumor activity and can be used for treating malignant tumor, apoplexy, paralysis and other difficult and complicated diseases. The fresh Gekko Swinhonis used in JINLONG Capsule is derived from Gekko Swinhonis without web. Due to partial consistency in habitat, such as feeding habits, aggregation on illuminated walls and the like, the species of the genus have convergent evolution on a certain character, so that the shape difference of various geckos is small. Unless a professional Chinese medicine identifier, it is difficult to distinguish various geckos from morphological features. Therefore, as a rare traditional Chinese medicine, the mixed and imitative product of gecko in the market is layered endlessly, and the administrator has weak identification capability on the qualified gecko and the gecko, so that a large amount of the gecko is sold as the gecko to gain violence.
At present, gecko is not loaded into Chinese pharmacopoeia, and researches on the gecko mainly comprise morphological identification, and the gecko is difficult to play a role in quality control of the preparation. The Gekko Swinhonis in Jinlong capsule is chemically characterized by finger print, and endogenous biological micromolecular substances such as uracil, hypoxanthine, xanthine, thymine and the like are detected. However, animal medicine has complex chemical components, the difficulty of separating and analyzing the compound is high, no special chemical components exist, the specificity of the detection method is low, and the qualitative and quantitative detection is difficult to accurately perform.
The DNA molecular technology is not affected by the form, chemical composition and the like of the sample, and can accurately determine the biological origin. Wherein, the fluorescence quantitative PCR method (Quantitative Real-time PCR, qPCR) and the microdroplet Digital PCR method (ddPCR) can be used for qualitative detection, judging the specificity of the primer and realizing the quantitative detection of the target fragment. In the previous research of the applicant, the copy number of the target fragment is found to have a certain correlation with the original feeding amount, and the DNA copy number or the license can become a new index for controlling the quality of the Chinese patent medicine.
Therefore, the invention combines the two technologies, and then performs quality control on the gecko component in the Jinlong capsule by calculating and analyzing the copy number, thereby establishing an accurate and effective molecular quantification method and providing a new idea for the quality control of the Jinlong capsule.
Disclosure of Invention
Aiming at the technical problems, the invention provides a quality control method with high accuracy, good specificity, high sensitivity and good repeatability, which realizes qualitative and quantitative detection of the webless gecko component in the Jinlong capsule.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a primer and a probe for detecting a webless gecko, wherein the primer comprises a primer 1 and a primer 2, the sequence of the primer 1 is shown as SEQ ID NO. 1, and the sequence of the primer 2 is shown as SEQ ID NO. 2; the sequence of the probe is shown as SEQ ID NO. 3.
The second aspect of the invention provides a PCR identification method for detecting a webless gecko, comprising the following steps:
extracting DNA in a target detection object; according to the designed and synthesized webless gecko specific primers and TaqMan specific probes; qPCR detection is carried out;
or extracting DNA in the target detection object; according to the designed and synthesized webless gecko specific primers and TaqMan specific probes; performing ddPCR detection;
the nucleotide sequence of the TaqMan specific probe is shown as SEQ ID NO. 3.
Further, the webless gecko-specific primers comprise a primer 1 and a primer 2; wherein, the sequence of the primer 1 is shown as SEQ ID NO. 1, and the sequence of the primer 2 is shown as SEQ ID NO. 2.
Further, the PCR identification method for detecting the webless geckos specifically comprises the following steps:
extracting DNA in a target detection object; according to the webless gecko specific primers and the TaqMan specific probes; qPCR detection is carried out;
or extracting DNA in the target detection object; according to the webless gecko specific primers and the TaqMan specific probes; performing ddPCR detection;
the nucleotide sequence of the TaqMan specific probe is shown as SEQ ID NO. 3; the webless gecko specific primer comprises a primer 1 and a primer 2; wherein, the sequence of the primer 1 is shown as SEQ ID NO. 1, and the sequence of the primer 2 is shown as SEQ ID NO. 2.
In the technical scheme of the invention, the judging conditions of the qPCR identification method for detecting the webless gecko are as follows: when the Ct value is less than or equal to 35, the result is positive, namely the target detection object comprises the webless gecko, and when the Ct value is greater than 35, the result is negative, namely the target detection object does not comprise the webless gecko.
In the technical scheme of the invention, the judging conditions of the ddPCR identification method for detecting the webless gecko are as follows: judging according to the detected positive droplets, wherein when a large number of positive droplets appear, the target detection object comprises the webless gecko, and when a large number of positive droplets do not appear, the target detection object does not comprise the webless gecko.
The third aspect of the invention provides the application of the primer and the probe for detecting the webless gecko or the PCR identification method for detecting the webless gecko in qualitative detection of the gecko component of the Jinlong capsule.
The fourth aspect of the invention provides a quality control method for detecting the gecko component of the Jinlong capsule based on a molecular quantification technology, which comprises the following steps:
designing a synthetic species specific primer and a specific probe according to the mitochondrial genome sequence difference of the gecko original animal webless gecko and the mixed imitative product; extracting DNA of qualified products of the Jinlong capsules, and determining the copy number range of the specific fragments of the gecko without web in the qualified products of the Jinlong capsules through qPCR; extracting DNA of a gold dragon capsule sample to be detected, performing qPCR, and calculating the copy number of the specific fragment of the webless gecko; comparing with qualified products;
or, designing a synthetic species specific primer and a specific probe according to the sequence difference of mitochondrial genome of the gecko original animal webless gecko and the mixed and imitative animal; extracting DNA of qualified products of the Jinlong capsules, and determining the copy number range of the specific fragments of the gecko without web in the qualified products of the Jinlong capsules through ddPCR; extracting DNA of a gold dragon capsule sample to be detected, performing ddPCR, and calculating the copy number of the specific fragment of the webless gecko; comparing with qualified products;
wherein the sequence of the specific probe is shown as SEQ ID NO. 3;
preferably, the 5 'end of the specific probe is modified with a fluorescent group 6-FAM, and the 3' end is modified with a fluorescence quenching group BHQ1.
Further, the quality control method for detecting the gecko components of the Jinlong capsules based on the molecular quantification technology specifically comprises the following steps:
designing and synthesizing a webless gecko specific primer and a TaqMan specific probe; extracting DNA in the qualified golden dragon capsules and determining the copy number range of the Gekko Swinhonis specific fragments in the qualified golden dragon capsules through qPCR; extracting DNA in the Jinlong capsules to be detected, performing qPCR detection, and calculating the copy number of the Gekko Swinhonis specific fragments; comparing;
the sequence of the TaqMan specific probe is shown as SEQ ID NO. 3;
preferably, the TaqMan specific probe is modified with a fluorescent group 6-FAM at the 5 'end and a fluorescent quenching group BHQ1 at the 3' end.
Or, specifically, the method comprises the following steps: designing and synthesizing a webless gecko specific primer and a TaqMan specific probe; extracting DNA in the qualified golden dragon capsules and determining the copy number range of the Gekko Swinhonis specific fragments in the qualified golden dragon capsules through ddPCR; extracting DNA in the Jinlong capsule to be detected, performing ddPCR detection, and calculating the copy number of the specific fragment of the Gekko Swinhonis; comparing;
the sequence of the TaqMan specific probe is shown as SEQ ID NO. 3;
preferably, the TaqMan specific probe is modified with a fluorescent group 6-FAM at the 5 'end and a fluorescent quenching group BHQ1 at the 3' end.
As a preferred embodiment, the copy number of the Gekko Swinhonis specific fragment in the qualified product of the qPCR-confirmed Jinlong capsule is not less than 2×10 2 Copies/mg, preferably 2X 10 2 ～5×10 2 The copies/mg, more preferably 2.39X10 2 ～4.60×10 2 copies/mg；
The copy number of Gekko Swinhonis specific fragment in qualified product of the ddPCR confirmed JINLONG Capsule is not less than 2×10 2 Copies/mg, preferably 2X 10 2 ～5×10 2 The copies/mg, more preferably 2.29X 10 2 ～4.54×10 2 copies/mg。
In the technical scheme of the invention, the applicant uses gold dragon capsules of different batches collected in the market as qualified product templates, determines that the copy number of the specific fragments of the Gekko Swinhonis is within a certain range through qPCR or ddPCR, has consistency, reflects the relation of the feeding amount according to the copy number, and is used for judging whether the Gekko Swinhonis content in the gold dragon capsules to be detected is qualified or not by calculating the copy number of the specific fragments of the Gekko gecko of the gold dragon capsules to be detected and comparing the copy number with the copy number of the specific fragments of the Gekko gecko of the qualified products qPCR or ddPCR so as to realize the quality control of the Gekko gecko components in the gold dragon capsules.
Further, the webless gecko-specific primers comprise a primer 1 and a primer 2; the sequence of the primer 1 is shown as SEQ ID NO. 1, and the sequence of the primer 2 is shown as SEQ ID NO. 2.
Further, the method for extracting the DNA in the gold dragon capsules to be detected or the qualified gold dragon capsules comprises the following steps:
(1) Taking the content of the capsule, adding buffer solution I, adding polyvinylpyrrolidone 40 (PVP 40) and beta-mercaptoethanol, carrying out water bath at 65 ℃ for 2 hours, shaking for 4-10 times during the water bath, and centrifuging to obtain supernatant;
(2) Adding methanol with the volume 1-2 times of that of the supernatant, standing at-20 ℃ for precipitation for at least 1h, centrifuging, discarding the supernatant, and reserving the precipitation;
(3) Adding buffer solution II and beta-mercaptoethanol into the precipitate, carrying out water bath at 65 ℃, shaking and uniformly mixing to dissolve the precipitate;
(4) Adding an equal volume of chloroform/isoamyl alcohol mixed solution, uniformly mixing, centrifuging, and taking supernatant;
(5) Adding 1-2 times of precooled absolute ethanol and one tenth of 3M NaAc solution of the supernatant, and settling at-20deg.C for at least 1 hr;
(6) Transferring the liquid and the precipitate obtained in the step (5) into a DNA adsorption column, centrifuging, and discarding the liquid;
(7) Adding buffer solution GD into the DNA adsorption column, centrifuging, and discarding the liquid; adding a rinsing liquid PW, centrifuging and discarding the liquid;
(8) Adding an elution buffer TE into the DNA adsorption column, centrifuging and completing the collection of DNA.
Wherein the buffer solution I in the step (1) comprises a buffer solution A and a buffer solution B; the buffer solution A consists of CTAB cell lysate, tris-HCl buffer solution, EDTA and NaCl; the buffer solution B is a DTT solution;
the buffer solution II in the step (2) is buffer solution A.
In the technical scheme of the invention, the DNA in the Jinlong capsule is extracted by combining methanol with an adsorption column, so that adverse effects of a preparation process and auxiliary materials such as starch and the like on DNA extraction in the preparation process of the Jinlong capsule are effectively overcome, the primer is more easily combined with a template, and the amplification requirement of the primer is met, thereby improving the purity of the DNA. In the preparation process of the Jinlong capsule, the proportion of the added auxiliary materials such as beta-cyclodextrin, starch and the like in the capsule is about 68.8 percent, the chemical and physical properties of the beta-cyclodextrin are stable, DNA can be embedded, and the difficulty is added to DNA extraction. In the technical scheme of the invention, the concentration of CTAB is increased to optimize the extraction formula, and the digestion capacity is improved by adding DTT and PVP40, so that the influence of beta-cyclodextrin encapsulation and starch covering is reduced. And the DTT is added into the DNA solution and used as a reducing agent and a deprotection agent of the sulfhydrylation DNA, thereby being beneficial to DNA release. While PVP40 removes phenolic and polysaccharide impurities.
If not further treated by the adsorption column, a large amount of white insoluble impurities are generated, and the substance, namely beta-cyclodextrin, is suspected to have a great influence on the subsequent PCR amplification. Subsequent PCR amplification may fail without treatment by the adsorption column.
Further, in the buffer solution A, the concentration of CTAB is 40g/L, the concentration of Tris-HCl is 0.1M, the concentration of EDTA is 0.02M, and the concentration of NaCl is 1.4M; the buffer B is a 1M DTT solution;
preferably, in the step (1), the buffer solution I is used in an amount of 3-4 mu L of buffer solution A and two thousandths of buffer solution B of the volume of the buffer solution A for each 1mg of the gold dragon capsule content; the addition amount of the polyvinylpyrrolidone 40 is 125g of polyvinylpyrrolidone 40 added into each 2.5mL of buffer solution A; the addition amount of the beta-mercaptoethanol is 100 mu L of beta-mercaptoethanol added into each 2.5mL of buffer solution A;
preferably, in step (3), buffer II is used in an amount of 600 μl of buffer a per 700mg of the contents of the gold dragon capsule; the amount of beta-mercaptoethanol added was 2. Mu.L of beta-mercaptoethanol per 75. Mu.L of buffer II.
The technical scheme has the following advantages or beneficial effects:
the invention designs and synthesizes a specific primer of the webless gecko and a Taqman probe based on the COI fragment, and establishes an identification method for reflecting the actual feeding amount of the webless gecko in the gold dragon capsule by the copy number through qPCR and ddPCR technologies.
The invention has the following advantages:
(1) According to the invention, a specific probe is analyzed and designed by utilizing a gene informatics technology, and a Taqman probe is utilized to qualitatively detect whether the gold dragon capsule product contains the webbed gecko component or not efficiently, accurately and rapidly, and the content analysis of the webbed gecko in the gold dragon capsule product is realized by comparing the copy number with the copy number range of qualified gold dragon capsule products;
(2) The combination of the primer and the probe used in the invention has strong specificity and good sensitivity, and can effectively realize quantitative detection of the webless gecko animal source component;
(3) The invention adopts the method of combining methanol with an adsorption column to extract DNA in the Jinlong capsule, improves the quality of DNA purification, reduces the influence of components such as sugar and the like on PCR amplification, and ensures that the identification result is more reliable and stable.
(4) The detection method provided by the invention has the advantages of good repeatability, strong specificity, high accuracy, large universal quantity and convenience in use, and has higher application value in monitoring and supervising the production and processing quality of the Jinlong capsules.
Drawings
FIG. 1 shows the results of a quantitative qPCR detection of the specificity of the primer probe of the webless gecko sample in example 1.
FIG. 2 shows the result of the detection of the specificity of the primer probe of the webless gecko sample by ddPCR in example 2.
FIG. 3 shows the result of a gradient sensitivity experiment for quantitative qPCR detection of a webless gecko sample in example 1.
FIG. 4 is a standard curve for qPCR quantitative detection of webless gecko samples in example 1.
FIG. 5 shows the result of a gradient sensitivity experiment for quantitative detection of a webless gecko sample by ddPCR in example 2.
FIG. 6 is a linear range of ddPCR quantitative detection of webless gecko samples in example 2.
FIG. 7 is a linear relationship between the amount of gecko fed and the DNA copy number (Lg 10) in the self-made test sample in example 3.
FIG. 8 is a Bland-Altman plot of quantitative detection of webbed gecko components by qPCR and ddPCR in the commercial gold dragon capsules of example 6.
Detailed Description
The following examples are only some, but not all, of the examples of the invention. Accordingly, the detailed description of the embodiments of the invention provided below is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to fall within the scope of the present invention.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the description of the embodiments is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the invention. Unless otherwise specifically indicated, the various materials, reagents, instruments and equipment used in the following examples of the invention are commercially available or may be prepared by existing methods.
1. Experimental materials
Gecko: webless gecko sample 3 parts A 1 -A 3 Wall of mixed and false productTiger samples total 5 parts A 4 -A 8 The species and sources of the 6 species of the family 2 are shown in Table 1.
Table 1 sample information table of webless gecko and mixed-fake gecko
Jinlong capsule: the commercial Jinlong capsules are all produced by Beijing Jian medical company, and have batch numbers of 160522, 161253, 190203, 190204, 190629, 190930, 190931, 190933, 190934 and 190937.
2. Specific primers and probes
The COI sequences of webless geckos and related pseudo products are aligned in molecular evolution genetic analysis software MEGA7.0, specific identification Primer pairs are designed by analyzing inter-species variation sites, and TaqMan specific probes are designed by using Primer Premier 5.0 software and Beacon Designer 7 software.
Primer Gswf129:5'-ATGTCGCAGTACAATACGCCC-3', SEQ ID NO. 1;
primer Gswr129:5'-TGTAGTGTTAAGGTTGCGGTCAGT-3', SEQ ID NO. 2;
the probe is Gspro:5'-TACCGCCGTACTACTCTTACTTGC-3', SEQ ID NO 3;
the probe Gspro is modified by a fluorescent group and a fluorescence quenching group; wherein, the 5 '-end modifies the fluorescent group 6-FAM and the 3' -end modifies the fluorescence quenching group BHQ1. Primers and probes were provided by Shanghai and Instroma respectively.
3. DNA extraction
Taking preserved gecko A without web 1 -A 3 Gekko Swinhonis A as a mixed and pseudo product 4 -A 8 The muscle tissue of the specimen is about 30mg, the tissue is sequentially cleaned, wiped and dried by ethanol with the mass fraction of 75% and deionized water, then the tissue is sheared by sterilized scissors, and DNA is extracted by using a root blood/cell/tissue genome DNA extraction kit.
About 700mg of commercial gold dragon capsule powder of different batches was taken and DNA was extracted using a methanol precipitation combined with adsorption column method.
4. qPCR amplification
qPCR amplification system: 2. Mu.L of DNA template; the concentration of each of the upstream and downstream primers (i.e., specific primers) was 0.4. Mu.L (final concentration: 200 nmol/L); probe (Gspro) 0.2. Mu.L (final concentration 100 nmol/L); bester qPCR MasterMix 10 μL; quickCut TM HindIII 1. Mu.L; 6. Mu.L of deionized water and a total reaction volume of 20. Mu.L. After reagent preparation is completed, the reagent is centrifuged briefly to eliminate bubbles in the octant tube, and the influence of the bubbles on fluorescence signal collection is reduced. Experiment in96 real-time fluorescent quantitative PCR instrument (Roche, switzerland). The amplification was performed using a two-step method, qPCR amplification procedure: pre-denaturation at 95 ℃ for 120s; denaturation at 95℃for 10s and annealing at 60℃for 30s for 40 cycles, and fluorescence signals were collected at the end of extension of each cycle. A blank control group without any species DNA template added (double distilled water substitution) was set up in the experiment.
5. ddPCR amplification
ddPCR amplification System: 2. Mu.L of DNA template; 1.8 mu L of each of the upstream and downstream primers, and the final concentration is 10 mu mol/L; probe Gspro 0.5. Mu.L with a final concentration of 250nmol/L; ddPCR TM Supermix for Probes 10μL；QuickCut TM HindIII fast-cutting enzyme 1. Mu.L; 2.9. Mu.L of deionized water and a total reaction volume of 20. Mu.L. Experiments were performed on a QX200 Droplet Digital PCR instrument (Bio-Rad, U.S.A.). The ddPCR amplification procedure was: 95 ℃ for 10min;94 ℃ for 30s,60 ℃ for 60s and 40 cycles; and 98 ℃ for 10min. A blank control group without any species DNA template added (double distilled water substitution) was set up in the experiment.
6. Specificity evaluation experiment of PCR primer and probe
Respectively adding a webless gecko sample A by using a specific primer and a probe 1 -A 3 Gekko Swinhonis sample A as a mixed and pseudo product 4 -A 8 The DNA template of (2) was subjected to PCR reaction as described above, and the specificity of the designed primers and probes was examined.
7. Linear range and sensitivity evaluation experiments of detection methods
Plasmid standards were supplied by saint (beijing) biotechnology development limited. Standards were subjected to qPCR and ddPCR assays, respectively, at 10-fold gradient dilutions, to evaluate the linear range and sensitivity of both methods.
8. Stability, repeatability and recovery evaluation experiments of the detection methods
Taking 1 part of DNA of the same batch of commercial Jinlong capsules, respectively carrying out independent repeated experiments of qPCR and ddPCR for 6 times in different time periods of 0, 2, 4, 8, 12 and 24 hours, and analyzing differences in difference groups among groups to verify the stability of the method.
In order to test the repeatability, 6 parts of commercial gold dragon capsules of the same batch number are also taken, the DNA is extracted according to the method, then qPCR and ddPCR reactions are respectively carried out, 3 duplicate wells are carried out on each sample in the same test, and the relative standard deviation RSD value of 6 batches of samples is calculated.
Taking the copy number measured by a standard substance with the dilution concentration of 0.11 ng/. Mu.L as a theoretical value, adopting a blank labeling recovery method, namely taking the standard substance for DNA extraction, finally adding ultrapure water with the same volume for dissolution, respectively carrying out qPCR and ddPCR amplification on the obtained DNA sample, taking the measured copy number as a value to be measured, and finally calculating the recovery rate (the value to be measured/the theoretical value multiplied by 100%).
The technical scheme of the invention is specifically illustrated below by combining specific embodiments:
example 1: qPCR quantitative detection gecko sample
Fluorescent quantitative PCR amplification system in this example: 2. Mu.L of DNA template; the concentration of each of the upstream and downstream primers (i.e., specific primers) was 0.4. Mu.L (final concentration: 200 nmol/L); probe (Gspro) 0.2. Mu.L (final concentration 100 nmol/L); bester qPCR MasterMix 10 μL; quickCut TM HindIII 1. Mu.L; 6. Mu.L of deionized water and a total reaction volume of 20. Mu.L. After reagent preparation is completed, the reagent is centrifuged briefly to eliminate bubbles in the octant tube, and the influence of the bubbles on fluorescence signal collection is reduced. Experiment in96 real-time fluorescent quantitative PCR instrument (Roche, switzerland). The amplification was performed using a two-step method, qPCR amplification procedure: pre-denaturation at 95 ℃ for 120s; denaturation at 95℃for 10s, annealing at 60℃for 30s, total 40 cycles, at each timeFluorescence signals were collected at the end of each cycle extension. A blank control group without any species DNA template added (double distilled water substitution) was set up in the experiment.
Specificity (one)
Respectively adding a webless gecko sample A by using a specific primer and a probe 1 -A 3 Mixed-fake gecko sample A 4 -A 8 qPCR was performed on the DNA template of (C) and the specificity of the designed primers and probes was examined.
Wherein, the gecko A without web 1 -A 3 And its mixed and fake gecko A 4 -A 8 The qPCR amplification curve of (2) is shown in figure 1, 3 authentic webless gecko samples show obvious specific S-shaped fluorescence amplification curves, and Ct values are between 16.79 and 18.53, so that positive reaction is realized. Blank and 5 pseudo-products (A) 4 -A 8 ) Or no specific amplification curve or Ct value greater than 35 appears, and negative reaction is generated. The designed primer and probe have strong specificity and specificity.
(II) linearity and sensitivity
In qPCR, linear regression analysis was performed on the logarithm of the standard copy number (base 10, i.e. lg N) as the abscissa and the corresponding Ct value as the ordinate.
The concentration of the plasmid standard substance is 110 ng/. Mu.L measured by a Nanodrop 2000c ultra-micro ultraviolet spectrophotometer, and the initial copy number concentration of the standard substance is calculated to be 3.42 multiplied by 10 10 COPies/. Mu.L. In qPCR, as shown in FIG. 3, the standard was diluted 10 1 ～10 10 The times respectively correspond to 3.42 multiplied by 10 9 ～3.42×10 0 Until the standard is diluted 10 10 Multiple to 3.42×10 0 Amplification signal still remained at copy number concentration of copies/. Mu.L, average Ct value was 36.37, indicating high sensitivity of the method, NTC in FIG. 3 was blank.
To prevent interference of the mix amplification signal, LOD and LOQ of the qPCR method were set to 34.2 copies/. Mu.L. As shown in FIG. 4, the method was carried out at a template concentration of 3.42X 10 1 ～3.42×10 9 The linearity in the range of copies/. Mu.L is good, and the regression equation of the standard curve is: y= -3.2393x+37.971, correlation coefficient R 2 =0.9997, primerThe amplification efficiency reaches 103.6%.
Example 2: ddPCR quantitative detection gecko sample
In this embodiment: microdroplet digital PCR amplification system: 2. Mu.L of DNA template; 1.8 mu L of each of the upstream and downstream primers, and the final concentration is 10 mu mol/L; probe Gspro 0.5. Mu.L with a final concentration of 250nmol/L; ddPCR TM Supermix for Probes 10μL；QuickCut TM HindIII fast-cutting enzyme 1. Mu.L; 2.9. Mu.L of deionized water and a total reaction volume of 20. Mu.L. Experiments were performed on a QX200 Droplet Digital PCR instrument (Bio-Rad, U.S.A.). The ddPCR amplification procedure was: 95 ℃ for 10min;94 ℃ for 30s,60 ℃ for 60s and 40 cycles; and 98 ℃ for 10min. A blank control group without any species DNA template added (double distilled water substitution) was set up in the experiment.
Specificity (one)
Respectively adding a webless gecko sample A by using a specific primer and a probe 1 -A 3 Mixed-fake gecko sample A 4 -A 8 ddPCR was performed on the DNA template of (C) to examine the specificity of the designed primers and probes.
Wherein, the gecko A without web 1 -A 3 And its mixed and fake gecko A 4 -A 8 The ddPCR detection results of (2) are shown in FIG. 2, except for A 1 -A 3 The webless gecko sample detects a large number of positive microdroplets, and the mixed product gecko A 4 -A 8 The sample showed few positive droplets. This shows that the primers and probes designed have very strong specificity and specificity.
(II) linearity and sensitivity
Plasmid standards in this example were provided by saint (beijing) biotechnology development limited. Standards were subjected to ddPCR assays at 10-fold gradient dilutions, assessing the linear range and sensitivity of the method.
In ddPCR assay, copy number was positively correlated with genomic DNA content (FIG. 5), log10 (copy number) was linearly correlated with Log10 (DNA content) (FIG. 6), and template concentration was 1.2X10 1 -2.35×10 4 The linearity in the range of copies/. Mu.L is good, R 2 The lowest limit of detection by ddPCR method was 0.79 copies/. Mu.L and the limit of quantification was 12 copies/. Mu.L.
Example 3: quantitative detection of webless gecko component in self-made test sample
In the embodiment, according to the golden dragon capsule, the mass ratio of Gekko Swinhonis to Agkistrodon is 2:1:1, and under the condition of keeping the total amount (60 mg) unchanged, an adulteration model with known mixing ratio of the webless gecko and the exendin is established, and a test product is prepared. The sample prepared in this example was prepared by mixing Gekko Swinhonis and its mixed product, bungarus Parvus, and Agkistrodon at mass of 30mg, 15mg, and 15mg, respectively.
The test samples were divided into 4 groups in the experiment, as shown in table 2: the quality control method comprises the steps of respectively obtaining all-genuine groups A1-A3, positive-fake quality 1:1 groups B1-B3, positive-fake quality 1:4 groups C1-C3 and all-fake quality group D1.
TABLE 2 adulteration model of gold dragon capsule without web gecko component
In this example, the 4 sets of extracted DNAs were amplified by the qPCR and ddPCR methods described above using the extracted DNAs as templates, and the copy numbers were analyzed, and the results are shown in Table 3.
TABLE 3 quantitative determination results of webless gecko in self-made samples
As can be seen from Table 3, the inter-group CV values of the test samples of each group were [ ]<25%) and RSD value [ ]<3%) meets the requirements, which indicates that the DNA extraction method is stable. In qPCR, when the feeding amount of the webless gecko in the test sample is a full-quality product, the average copy number of the gecko is 1.75X10 6 cobies/mg; when the feeding ratio of the normal and the false products is 1:1, the average copy number is 8.11 multiplied by 10 5 cobies/mg; when the feeding ratio of the normal and the false products is 1:4, the average copy number is 3.13 multiplied by 10 5 copies/mg; in ddPCR, when the feeding amount of the webless gecko in the test sample is a full-quality product, the average copy number is 9.95 multiplied by 10 5 cobies/. Mu.L; when the feeding ratio of the normal and the false products is 1:1, the average copy number is 4.64 multiplied by 10 5 cobies/. Mu.L; when the feeding ratio of the normal and the false products is 1:4, the average copy number is 1.97X10 5 COPies/. Mu.L. No obvious positive microdrops appear in the whole pseudo product group and the blank control group without the webless gecko. The actual feeding ratio of the webless gecko in the test sample is about 10.3:5:2, ratio of actual copy number measured by qpcr is 10:4.6:1.8, the ratio of the actual copy numbers measured in ddPCR was 10:4.7:2, the ratio of the actual feed ratio to the actual copy number of the two processes is substantially identical. As shown in fig. 7, the weight of the webless gecko is proportional to Lg10 (copy number), indicating that the copy number can reflect the actual dosage, and both quantitative methods can be used to estimate the original dosage.
Thus, the actual dosage is reflected by an analysis of whether the ratio of the dosage to the copy number is consistent or not, and can be used as a reference for the original dosage.
Example 4: extraction of DNA from commercial gold dragon capsules
In this example, the procedure for extracting DNA from Jinlong capsules was as follows:
(1) Preparing a buffer solution A: 1.0M Tris-HCl (pH 8.0) 10mL,5.0M NaCl solution 28mL,0.5M EDTA solution (pH 8.0) 4mL and 4g CTAB are taken, mixed and added with water to fix the volume to 100mL;
(2) 1.0M buffer B DTT solution was prepared: 1.54g of DTT was weighed and dissolved in 10mL of ddH 2 Packaging in a centrifuge tube of 1.5mL, and preserving at-20deg.C;
(3) Taking 700mg of golden dragon capsule content in a 7mL centrifuge tube; 2.5mL of preheated buffer A (5 mu L of buffer B, namely two thousandths of the buffer B is added before preheating) is added, 125mg of PVP40 is added to make the final concentration of the buffer A be 5%, 100 mu L of beta-mercaptoethanol is added to make the final concentration of the buffer A be 4%, and vortex oscillation is carried out for 1min, so that the samples are fully and uniformly mixed;
(4) Water bath at 65 ℃ for 2 hours, and gently shaking and uniformly mixing every half an hour during the water bath;
(5) Taking out, cooling on ice, and centrifuging at 12000r/min for 15min;
(6) Taking supernatant, split charging the supernatant into two 7mL centrifuge tubes on average, adding 2.5mL of methanol respectively, and precipitating for 1h at-20 ℃;
(7) Centrifuging at 12000r/min for 5min, and combining the precipitates of the two centrifuge tubes into one 7mL centrifuge tube; 600. Mu.L of preheated buffer A and 16. Mu.L of mercaptoethanol are added into the precipitate, and the mixture is placed at 65 ℃ for cracking for 20min, and the mixture is gently shaken and mixed to dissolve the precipitate;
(8) Transferring the solution obtained in the previous step into a 2mL centrifuge tube, adding an equal volume of chloroform/isoamyl alcohol (volume ratio is 24:1) mixed solution, gently shaking and mixing for 5min, centrifuging for 15min at 12000r/min, and taking supernatant;
(9) Adding the supernatant, the equal volume of precooled absolute ethyl alcohol and one tenth volume of 3M NaAc solution of the supernatant, and settling at-20 ℃ for at least 1h;
(10) Placing the adsorption column CB3 into a collecting pipe, collecting the liquid and the sediment obtained in the previous step into the adsorption column for multiple times, centrifuging for 30s at each time of 12000r/min, discarding the liquid, and placing the adsorption column into the collecting pipe;
(11) Adding 500 μl of buffer GD, centrifuging at 12000r/min for 1min, discarding the liquid, and placing the adsorption column back into the collection tube;
(12) Adding 600 μl of rinse solution PW, centrifuging at 12000r/min for 1min, discarding the liquid, and placing the adsorption column back into the collection tube; repeating the step once;
(13) Centrifuging at 12000r/min for 2min to remove residual liquid in the adsorption column;
(14) Taking out the adsorption column, drying in a baking oven at 38 ℃ for 20min, taking out the adsorption column after drying, putting the adsorption column into an EP tube with 1.5mL, suspending and dripping 70 mu L of elution buffer TE in the middle of the adsorption column, standing at room temperature for 5min, and centrifuging for 2min at 12000r/min to finish DNA collection. Finally, the collected DNA was stored in a-20℃refrigerator.
Example 5: qPCR quantitative detection of webless gecko component of commercial gold dragon capsule
Taking commercial golden dragon capsule samples of different batches, extracting DNA as a template according to the method, carrying out qPCR measurement, carrying out parallel measurement on each sample for three times, setting a blank control (double distilled water replaces the DNA template), and calculating copy number in units of copies/mg.
Stability, repeatability and recovery
In this example, commercial Jinlong capsules with lot number 190937 were taken and qPCR was performed 6 times in different time periods of 0, 2, 4, 8, 12, and 24 hours, respectively, to examine the repeatability and stability of the method.
In the stability investigation, the RSD value in the same sample group is 0.16% -0.60%, the RSD value between groups is 0.93%, and the RSD values are less than 3%, which shows that the extracted DNA of the commercial golden dragon capsule remains stable within 24 hours after repeated freeze thawing (see table 4). RSD values for 6 parallel samples ranged from 0.46% to 2.23% (see table 5). According to the analysis method of the 2020 edition of Chinese pharmacopoeia, the content is lower than 0.0001% (here, the content of standard substance in each 100ml solution is lower than 10) -7 g) The recovery limit was 75-120%, and the theoretical concentration was 3.42×10 7 The recovery rate of the copies/. Mu.L standard DNA is between 96.51% and 101.16% (see Table 6), meets the recovery rate limit requirement, and shows that the detection method has good accuracy.
Table 4 qPCR stability test of commercial gold dragon capsules lot number 190937 (n=3)
Table 5 qPCR reproducibility test of commercial gold dragon capsules lot number 190937 (n=3)
TABLE 6 qPCR blank sample recovery assay
(II) quantitative detection of Gekko Swinhonis component in commercial gold dragon capsule
The extracted DNA of 10 batches of gold dragon capsules is amplified by using an established qPCR method, and the final result is converted into units of copies/mg. The calculation formula of the copy number of the specific fragment of the webbed gecko contained in each milligram of the gold dragon capsule powder is shown as the formula (I):
the results are shown in Table 10 (the amount of the feed in Table 10 refers to the weight of the content of the gold dragon capsule used in extracting DNA from the gold dragon capsule), and the copy number of the specific fragment of the webbed gecko-free component in the commercial gold dragon capsule is 2.29×10 2 -4.60×10 2 Between copies/mg.
Example 6: ddPCR quantitative detection of webbed gecko component in commercial gold dragon capsule
Taking commercial golden dragon capsule samples of different batches, extracting DNA as a template according to the method, carrying out ddPCR measurement, carrying out parallel measurement on each sample for three times, setting a blank control (double distilled water replaces the DNA template), and calculating copy number in units of copies/mg.
Stability, repeatability and recovery
Methodology investigation was performed on the established webless gecko ddPCR method in this example. The commercial Jinlong capsules with batch number of 190930 are taken, 6 ddPCR detection is carried out respectively at different time periods of 0, 2, 4, 8, 12 and 24 hours, and the repeatability and the stability of the method are examined. As shown in tables 7 and 8, the CV values between groups were lower than 25% of the allowable error, indicating that the established ddPCR detection method had good reproducibility and stability. The theoretical copy number obtained by direct ddPCR detection is 96.5 copies/. Mu.L, and the recovery rate of 6 standard products after DNA extraction is 78.76% -93.92% (Table 9). According to the requirements of pharmacopoeia 2020, the detected 6 samples are all in the range of the recovery rate acceptable by the quantitative detection method, which shows that the established ddPCR method has good accuracy.
Table 7 ddPCR stability test of commercial gold dragon capsules lot No. 190930 (n=3)
Table 8 ddPCR repeatability test of commercial gold dragon capsules lot No. 190930 (n=3)
TABLE 9 microdroplet digital PCR blank sample recovery assay
(II) quantitative detection of Gekko Swinhonis component in commercial gold dragon capsule
The extracted DNA of 10 batches of gold dragon capsules was amplified using the established ddPCR method, and the final result was converted into units of copies/mg. The calculation formula of the copy number of the specific fragment of the webbed gecko contained in each milligram of the gold dragon capsule powder is shown as the formula (II):
in formula (II), copy number (copies/μL) Is the copy number per microliter (which is read by a microdroplet digital PCR readout) in the amplification system (20 microliters in this example).
The results are shown in Table 10 (the amount of the feed in Table 10 refers to the weight of the content of the gold dragon capsule used in extracting DNA from the gold dragon capsule), and the copy number of the specific fragment of the webbed gecko-free component in the commercial gold dragon capsule is 2.29×10 2 -4.60×10 2 Between copies/mg. The copy number concentration of the webbed gecko component in the 10 batches of commercial gold dragon capsules is more than 2 multiplied by 10 2 Copies/mg, indicating that the preparation process is stable, suggesting that 2X 10 2 copy number of samples of copies/mg was used as one of the detection criteria. 10 batches of detection results of ddPCR of commercially available golden dragon capsules,and comparing with the detection result of qPCR. Bland-Altman plots (see FIG. 8) were made for the quantification of the Gekko components of the commercial gold dragon capsules, with all the measured data being within a 95% confidence interval, meaning that the two quantification methods were well consistent. The detection results of the two methods are basically consistent, which shows that the established qPCR and ddPCR detection methods can accurately and effectively detect the gecko components in the commercial golden dragon capsules.
Table 10 qPCR method and ddPCR method for detecting content of commercial gold dragon capsule webless gecko
In summary, the qPCR method and the ddPCR method established by the invention take DNA copy number as quantitative indexes, have the advantages of accuracy, stability, good reproducibility and the like, and can be used as an important reference for controlling the quality of the webless gecko component of the golden dragon capsule.
It should be noted that, when the present invention relates to numerical ranges, any one of the two endpoints of each numerical range and any one of the two endpoints may be selected unless the present invention is otherwise stated. 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. It is therefore intended that the following claims be interpreted as including the 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 modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, and all equivalent changes made by the specification and drawings of the present invention, or direct or indirect application in other related technical fields, are included in the scope of the present invention.
SEQUENCE LISTING
<110> university of medical science in south China
<120> method for quality control of gold dragon capsules based on molecular quantification technology detection of Gekko Swinhonis specific fragment copy number, and primers and probes
<130> 2021
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> artificial sequence
<400> 1
atgtcgcagt acaatacgcc c 21
<210> 2
<211> 24
<212> DNA
<213> Synthesis
<400> 2
tgtagtgtta aggttgcggt cagt 24
<210> 3
<211> 24
<212> DNA
<213> Synthesis
<400> 3
taccgccgta ctactcttac ttgc 24
Claims (7)
1. The quality control method for detecting the gecko components of the Jinlong capsules based on the molecular quantification technology is characterized by comprising the following steps:
designing a synthetic species specific primer and a specific probe according to the mitochondrial genome sequence difference of the gecko original animal webless gecko and the mixed imitative product; extracting DNA of qualified products of the Jinlong capsules, and determining the copy number range of the specific fragments of the gecko without web in the qualified products of the Jinlong capsules through qPCR; extracting DNA of a gold dragon capsule sample to be detected, performing qPCR, and calculating the copy number of the specific fragment of the webless gecko; comparing with qualified products;
or, designing a synthetic species specific primer and a specific probe according to the sequence difference of mitochondrial genome of the gecko original animal webless gecko and the mixed and imitative animal; extracting DNA of qualified products of the Jinlong capsules, and determining the copy number range of the specific fragments of the gecko without web in the qualified products of the Jinlong capsules through ddPCR; extracting DNA of a gold dragon capsule sample to be detected, performing ddPCR, and calculating the copy number of the specific fragment of the webless gecko; comparing with qualified products;
wherein the sequence of the specific probe is shown as SEQ ID NO. 3; the specific primer comprises a primer 1 and a primer 2; the sequence of the primer 1 is shown as SEQ ID NO. 1, and the sequence of the primer 2 is shown as SEQ ID NO. 2;
the method for extracting the DNA in the gold dragon capsule sample to be detected or the gold dragon capsule qualified product comprises the following steps:
(1) Taking the content of the capsule, adding buffer solution I, adding polyvinylpyrrolidone 40 and beta-mercaptoethanol, carrying out water bath 2h at 65 ℃ for 4 times, centrifuging, and taking supernatant;
(2) Adding methanol with the volume 1-2 times of that of the supernatant, standing at-20 ℃ for precipitation for at least 1h, centrifuging, discarding the supernatant, and reserving the precipitation;
(3) Adding buffer solution II and beta-mercaptoethanol into the precipitate, carrying out water bath at 65 ℃, shaking and uniformly mixing to dissolve the precipitate;
(4) Adding the mixed solution of chloroform/isoamyl alcohol with equal volume, uniformly mixing, centrifuging, and taking supernatant;
(5) Adding the supernatant, the equal volume of precooled absolute ethyl alcohol and one tenth volume of 3M NaAc solution of the supernatant, and settling at-20 ℃ overnight;
(6) Transferring the liquid and the precipitate obtained in the step (5) into a DNA adsorption column, centrifuging, and discarding the liquid;
(7) Adding buffer solution GD into the DNA adsorption column, centrifuging, and discarding the liquid; adding a rinsing liquid PW, centrifuging and discarding the liquid;
(8) Adding TE buffer solution into the DNA adsorption column, centrifuging, and completing collection of DNA;
wherein the buffer solution I in the step (1) comprises a buffer solution A and a buffer solution B; the buffer solution A consists of CTAB cell lysate, tris-HCl buffer solution, EDTA and NaCl; the buffer solution B is a DTT solution;
the buffer solution II in the step (2) is buffer solution A.
2. The quality control method for detecting the gecko component of the Jinlong capsule based on the molecular quantification technology of claim 1, wherein the 5 'end of the specific probe is modified with a fluorescent group 6-FAM and the 3' end is modified with a fluorescence quenching group BHQ1.
3. The quality control method for detecting gecko components of Jinlongjiao nang based on molecular weight determination technology of claim 1, wherein the copy number range of gecko specific fragments in qualified Jinlongjiao nang products confirmed by qPCR is 2.39X10 2 ~ 4.60×10 2 copies/mg。
4. The quality control method for detecting gecko components of Jinlongjiao nang based on molecular weight determination technology of claim 1, wherein the copy number of gecko specific fragments in qualified Jinlongjiao nang products confirmed by ddPCR is in the range of 2.29×10 2 ~ 4.54×10 2 copies/mg。
5. The quality control method for detecting the gecko component of the Jinlongjiao nang based on the molecular weight distribution technology according to claim 1, wherein the concentration of CTAB in the buffer A is 40g/L, the concentration of Tris-HCl is 0.1M, the concentration of EDTA is 0.02M, and the concentration of NaCl is 1.4M; the buffer B was a DTT solution of 1M.
6. The quality control method for detecting the gecko components of the Jinlong capsules based on the molecular quantification technology according to claim 1, wherein in the step (1), the consumption of the buffer solution I is that each 1mg golden dragon capsule content corresponds to 3-4 mu L of the buffer solution A and the buffer solution B of two thousandths of the volume of the buffer solution A; the polyvinylpyrrolidone 40 is added into the buffer solution A with the addition amount of each 2.5 to mL, and 125 to g polyvinylpyrrolidone 40 is added into the buffer solution A; and adding 100 mu L of beta-mercaptoethanol into the buffer solution A with the addition amount of the beta-mercaptoethanol of every 2.5 mL.
7. The quality control method for detecting the gecko components of the Jinlong capsules based on the molecular quantification technology according to claim 1, wherein in the step (3), the using amount of the buffer solution II is 600 [ mu ] L of the buffer solution II corresponding to each 700mg of the content of the Jinlong capsules; and adding 2 mu L of beta-mercaptoethanol into each 75 mu L of buffer solution II.
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