CN108118095A - The method of quality control of long-nosed pit viper ingredient in a kind of detection Jinlong capsule - Google Patents

Abstract

The present invention provides a kind of method of quality control for detecting long-nosed pit viper ingredient in Jinlong capsule, and its step are as follows：Extract the DNA in Jinlong capsule sample；Design and synthesize Taqman probes；Establish Taqman qPCR standard curves；Carry out Taqman qPCR detections.The method of the present invention can accurately, reliably and efficiently identify the long-nosed pit viper ingredient in Jinlong capsule.

Description

A quality control method for detecting Viper components in Jinlong Capsules

technical field

The invention belongs to the field of molecular biology, and in particular relates to a quality control method for detecting Aggresis components in Jinlong Capsules by qPCR based on Taqman probes.

Background technique

Jinlong Capsule is the first anti-cancer fresh animal drug approved by the state. Its prescription is composed of fresh Shougong, fresh white snake and fresh Viper in a ratio of 2:1:1. , can inhibit tumor growth, inhibit postoperative local recurrence and distant metastasis, regulate the body's immunity, and be used for the treatment of primary liver cancer, lung cancer and other cancers and adjuvant chemotherapy and radiotherapy. Among them, Viper is a precious Chinese medicinal material, which has the effects of dispelling wind, dredging collaterals, and relieving spasm. In recent years, the medicinal value and economic value of snakes have increased significantly, and the social demand continues to rise. With the gradual scarcity of wild snake resources and high prices, there are many counterfeit and confused products in the medicinal material market, and the quality problems are serious. It is difficult to accurately identify them only based on their external morphological characteristics. In addition, due to the low reproductive capacity of snakes and the fragile ecological environment, poaching, smuggling and abuse have threatened the survival of many snakes, especially endangered snakes. Accurate identification of snakes can not only accurately identify genuine medicinal materials and counterfeit products, but also ensure the safety and effectiveness of medication, and is also the primary link in strengthening the protection and management of snake resources.

In the development process of traditional Chinese medicine, animal medicine is an important part of it. Due to the complex chemical composition of animal medicine itself, the separation and analysis of compounds is difficult, and it is slightly inferior to botanical medicine in terms of specific identification and content determination research. Based on the qualitative and quantitative analysis methods at the chemical composition level, only a few animal drugs have specific quality control methods. For example, cantharidin can be quantitatively analyzed and detected by the liquid chromatography method of colleges and universities. However, most animal medicines such as antelope horn, deer antler, and hippocampus are mainly based on identifying their authenticity, and have no unique chemical components, and the commonly used identification methods are not very specific. With the development of molecular biotechnology, for traditional Chinese medicines, especially animal medicines, molecular means can be used to achieve highly specific identification of unclear chemical components or no proprietary chemical components. The Chinese Pharmacopoeia 2015 edition uses a conventional PCR method for the identification of vipers. First, its DNA is extracted, and then specific primers are used to amplify the target fragment. This method has strong specificity and no false positives, but it can only be used for qualitative identification. However, due to the serious degradation and fragmentation of DNA in Jinlong Capsule products, it is difficult to successfully extract high-quality DNA by conventional extraction methods, so it is difficult to amplify the target fragment and cause false negatives. For the vast majority of animal traditional Chinese medicines, there is no effective, reliable and widely applicable quality control method at present.

Real-time fluorescent quantitative PCR (Quantitative Real-time PCR) is a method for detecting the total amount of products after each polymerase chain reaction (PCR) cycle with fluorescent chemicals in DNA amplification reactions. A method for qualitative and quantitative analysis of a specific DNA sequence in a sample to be tested by using an internal or external reference method. The method detects the progress of PCR in real time through the analysis of fluorescent signals. The probe method is a common method in qPCR, adding chemically modified oligonucleotide probes to the reaction system. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group, and the fluorescent signal cannot be detected. When PCR is amplified, the 5'-3' end exonuclease activity of Taq DNase will digest and degrade the probe, so that the reporter fluorescent group and the quencher fluorescent group are separated, and the fluorescent reporter group and the quencher group The energy transfer structure between them is destroyed, so that the fluorescence detection system can receive the fluorescence signal, that is, every time a DNA strand is amplified, a fluorescent molecule is formed, realizing the complete synchronization of the accumulation of the fluorescence signal and the formation of the PCR product.

Contents of the invention

The purpose of the present invention is to aim at the above technical problems to be solved, and to provide a new quality control method that is convenient, fast, accurate, specific, high in sensitivity, and good in repeatability by using molecular means, and can quantitatively detect Acupuncture in Jinlong Capsules. Snake ingredients.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for quantitatively detecting Viper components in Jinlong Capsules, the steps are as follows:

(1) extract the DNA in the Jinlong Capsule sample;

(2) Design and synthesize Taqman probes;

(3) Establish Taqman qPCR standard curve;

(4) Perform Taqman qPCR detection.

Preferably, in step (1), a modified SDS extraction method is used for DNA extraction. More preferably, the reagents for DNA extraction include buffer A, buffer B and buffer C, wherein buffer A includes 1M Tris-HCL, 5M NaCL, 0.5MEDTA and sodium dodecyl sulfate, and buffer B includes 3mol/L sodium acetate, buffer C includes 2mg/mL proteinase K.

On the other hand, the extraction operation of step (1) is as follows: take the contents of 2 Jinlong capsules, add 2ml of buffer A, 100 μL of buffer C, shake and mix well, bathe in water at 65°C for 1.5 hours, add 500 μL of buffer B, Extract 2 mL of chloroform-isoamyl alcohol with a composition ratio of 24:1, add an equal volume of isoamyl alcohol to the supernatant to precipitate at -20°C for 3 hours, centrifuge at 12,000 rpm for 20 min, wash the precipitate with 75% ethanol, dry it in the air, and add TE to dissolve it. Purify DNA.

Preferably, in step (2), a Taqman probe is designed and synthesized based on the Viper-specific primer, the 5' end of the Taqman probe is fluorescently modified with a reporter group, and the 3' end is modified with a quencher group. The reporter group can be one of FAM, HEX, TAMRA, ROX, and CY5, and the quenching group can be one of Dabcyl, BHQ1, and BHQ2. More preferably, the nucleotide sequence of the Taqman probe is: 5'-FAM-CATCCATCATCCACAT CTCCCG AGACGTGC-BHQ1-3' (SEQ ID NO.1). More preferably, the nucleotide sequence of the Viper specific primer is as follows: 5'-GGCAA TTCAC TACAC AGCCA ACATC AACT-3' (SEQ ID NO.2), DK2 (5'-CCATA GTCAG GTGGT TAGTG ATAC-3'( SEQ ID NO. 3).

Preferably, step (3) includes preparing a standard product, wherein the DNA extracted from Viper musculature is amplified with the Cytb sequence universal primer: Cytb1 (5'-TACCATGAGGACAAATATCATTCTG-3'(SEQ ID NO.4)); Cytb2 (5'-CCTCCTAGTTTGTTAGGGATTGATCG-3'(SEQ ID NO.5)); the reaction system is: 20 μL of PCR Taq premix enzyme, 2 μL of upstream and downstream primers with a final concentration of 200 nmol/L, 12 μL of deionized water, and 4 μL of template. The total volume is 40 μL; the reaction conditions are: 94°C for 5 minutes; 94°C for 30s, 53°C for 60s, and 72°C for 60s, a total of 35 cycles; 72°C for 7 minutes; the PCR product was purified with a DNA purification and recovery kit, and the purified Viper The Cytb fragment was used as a standard and identified by sequencing. The DNA concentration and purity of the standard was determined. The standard was diluted 10 times with deionized water, and the diluted standard was used as a template for real-time fluorescent quantitative PCR to establish a standard curve.

Preferably, in step (4), the reaction system is: DNA template 2 μL, probe 0.2 μL, upstream and downstream primers 0.4 μL each with a final concentration of 200 nmol/L, Bester qPCR MasterMix Taqman 10 μL, ROX 50×0.2 μL, deionized water 6.8 μL for a total reaction volume of 20 μL.

Preferably, in step (4), the PCR reaction program is: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 10s, annealing at 50°C for 30s, extension at 72°C for 30s, a total of 40 cycles, and collecting fluorescence signals at the end of each cycle of extension .

The invention discloses a real-time fluorescent quantitative PCR quantification detection method for the Viper component probe method in Jinlong Capsules, and its innovation lies in:

(1) The present invention creatively optimizes the traditional animal tissue DNA extraction method—SDS method, and overcomes the difficult and incomplete extraction problem due to DNA degradation caused by Jinlong capsules undergoing a series of processing methods;

(2) The present invention utilizes gene informatics technology to analyze and design specific probe, by drawing standard curve, utilizes Taqman probe, can efficiently, accurately and more quickly qualitatively detect whether in Jinlong Capsule product contains Aggresis composition, also can Quantitative analysis of Viper components in the DNA extract of Jinlong Capsule products. At the same time, it can analyze and identify other counterfeit snake components commonly used in adulteration in Jinlong Capsules, so that the DNA extracted from Jinlong Capsules is stable and of good quality, which can meet the qPCR experiment;

(3) The combination of primers and probes used in the present invention has strong specificity and good sensitivity, and can effectively realize the quantitative detection of Viper animal source components;

(4) The method of the present invention has good repeatability, strong specificity, high accuracy, large versatility, and is easy to use;

(5) The present invention has high application value in the production and processing quality monitoring and supervision of Jinlong Capsules.

Description of drawings

Fig. 1 is the experimental result of Aggression primer probe specificity.

Figure 2 is Viper standard curve.

Figure 3 is the gradient sensitivity experiment results of Viper.

Figure 4 shows the detection results of Viper DNA in commercially available Jinlong Capsules.

Detailed ways

The content of the present invention will be further described below in conjunction with specific examples, but it should not be construed as a limitation of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention except primers and probes are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and kits used in the present invention are commercially available.

In the following examples, the experimental materials and reagents used are as follows:

Experimental Materials:

Three fresh tissues from different individuals of krait, cobra, red chain snake, haystigma lancehead, white krait, crocodile, mouse snake, and viper Fresh snake tissues and different batches of Jinlong Capsules (Beijing Jiansheng Pharmaceutical Co., Ltd.) were purchased from Baozhilin Pharmacy in Guangzhou.

Reagents used:

Primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. 2×TaqMan MasterMix is DBI Bioscience brand.

Example 1

1. DNA extraction system

The DNA extraction of the sample was carried out using the improved SDS extraction method, and the required reagents included: buffer A (1M Tris-HCL; 5M NaCL; 0.5M EDTA; sodium dodecyl sulfate), buffer B (3mol/L sodium acetate ), buffer C (2mg/mL proteinase K). The steps are as follows: Take the contents of 2 Jinlong capsules, add 2ml of buffer A, 100μL of buffer C, shake and mix well, bathe in water at 65°C for 1.5h, add 500μL of buffer B, 2mL of chloroform-isoamyl alcohol (24:1) Extract, add an equal volume of isoamyl alcohol to the supernatant to precipitate at -20°C for 3 hours, centrifuge at 12,000 rpm for 20 minutes, wash the precipitate with 75% ethanol, dry in the air, add TE to dissolve, purify DNA with Tiangen Purification and Recovery Kit, and follow the PCR product Purification steps are carried out.

2. System and procedures

For the sample to be tested, 20 μl of the reaction system mixed with the probe primer was used for detection and analysis.

Probe Design

The present invention draws on the Viper-specific primers recorded in the 2015 edition of "Chinese Pharmacopoeia", and uses the specific primers DK1 (5'-GGCAA TTCAC TACAC AGCCAACATC AACT-3'), DK2 (5'-CCATA GTCAG GTGGT TAGTGATAC-3' ) to amplify the DNA of Viper and its counterfeit products. The Taqman probe was designed using premier primer 5.0, and the probe DKTZ (5'FAM-CATCCATCATCCACATCTCCCGAGACGTGC-BHQ13') was designed according to the specific primer DK1/DK2. The 5' end of the specific probe is fluorescently modified with a reporter group, and the 3' end is modified with a quencher group. The reporter group can be FAM, HEX, TAMRA, ROX, CY5, etc., and the quenching group can be Dabcyl, BHQ1, BHQ2, etc. The probes were analyzed again using Blast to ensure the specificity of the probes.

20 μl reaction system: 2 μL DNA template, 0.2 μL probe, 0.4 μL upstream and downstream primers (final concentration 200 nmol/L), 10 μL Bester qPCR MasterMix (Taqman Probe), 0.2 μL ROX (50×), 6.8 μL deionized water.

The PCR reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, a total of 40 cycles, and fluorescence signals were collected at the end of each cycle of extension.

3. Quantitative analysis

First, establish a Taqman qPCR standard curve: prepare standard products, and amplify DNA extracted from Viper musculature with Cytb sequence universal primers: Cytb1 (5'-TACCATGAGGACAAATATCATTCTG-3'); Cytb2 (5'-CCTCCTAGTTTGTTAGGGATTGATCG-3') , Reaction system: 20 μL of PCR Tap premix enzyme, 2 μL of upstream and downstream primers (final concentration 200 nmol/L), 12 μL of deionized water, 4 μL of template, and a total reaction volume of 40 μL; the reaction conditions are: 94 °C for 5 min; 94 °C for 30 s, 53°C for 60s, 72°C for 60s (35 cycles in total); 72°C for 7min. The PCR product was purified with a DNA purification and recovery kit. The purified Viper Cytb fragment was used as a standard product and identified by sequencing. The DNA concentration and purity of the standard product were measured using a NanoDrop One ultra-micro-volume ultraviolet spectrophotometer. Calculate the copy number: N(copies/μL)=(6.02×10 ²³ )×(ng/μL×10 ^-9 )/(DNA length×660). The standard was serially diluted 10 times with deionized water, and the diluted standard was used as a template for real-time fluorescent quantitative PCR to establish a standard curve.

Extract 5 different batches of Jinlong Capsule DNA, carry out real-time fluorescent quantitative PCR, and carry out qPCR according to the above-mentioned qPCR reaction system and procedure, set three replicate samples for each concentration point, use the drawn standard curve to analyze the unknown samples. The concentration of Viper DNA template was determined.

The steps of Taqman qPCR specific experiment among the present invention are as follows:

Using specific primers and probes, add 9 species of golden krait, cobra, red chain snake, haystigma lancehead, white krait black back, Elaphe japonica, Elaphe japonica, mouse snake, and viper rotundus, respectively. The DNA template of a common adulterated snake was used for fluorescence specific experiments, and the reaction system was established according to the optimal reaction system determined by the fluorescent quantitative PCR detection method.

The steps of the sensitivity experiment of Taqman qPCR among the present invention are as follows:

Dilute the standard stock solution according to a 10-fold gradient to obtain 100, ^{10 -1} , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 -5 , 10 ^-6 , ^{10 -7 ,} 10 ^-8 , 10 ^{- 9x} DNA stock solution. Detection with specific primers and probes.

The steps of the repetitive experiment of Taqman qPCR among the present invention are as follows:

Six different concentrations of standard DNA were used for repeated detection with specific primers and probes, and the relative standard deviation (RSD) of Ct values within and between experiments was calculated to verify the precision of the method.

The steps of the accuracy experiment of Taqman qPCR among the present invention are as follows:

The average Ct value of the standard DNA detected by Taqman probe method fluorescent quantitative PCR for more than 6 times was set as the theoretical value, an equal volume of standard solution was added to the blank matrix DNA extraction solution, and the Taqman probe was carried out by using the blank standard addition recovery method. Fluorescence quantitative PCR amplification by needle method, and the recovery rate was calculated (measured value/theoretical value×100%).

4. Test results

The test results are shown in Figure 1 to Figure 4. Wherein, Fig. 1 is the experimental result of Viper primer probe specificity. The test results of 10 different species showed that 3 vipers from different individuals had a typical "S" amplification curve, while the other 9 counterfeit snakes and the blank control had no amplification curve or Ct value greater than 38 . Figure 2 is Viper standard curve. Figure 3 shows the results of the gradient sensitivity experiment of Viper, which shows that when the standard product is diluted to 2.454×10 ² copies/μL, there is an obvious amplification signal, and the Ct value is 39.72. Considering that the counterfeit product also has an amplification signal at this Ct value, It is considered that the minimum detection of this method is 2.454×10 ³ copies/μl, and the Ct value is 37.76. Figure 4 shows the detection results of Viper DNA in commercially available Jinlong Capsules. The results in Figure 4 show that the DNA extract of the unknown Jinlong Capsule product, after qPCR amplification, has an obvious amplification curve of Viper, indicating that it is Viper. The concentration was determined by Viper standard curve, and the result was 2.5×10 ⁴ copies/μshell to 2.5×10 ⁶ copies/μshell. The test results show that this method is accurate, reliable and effective for the identification of Viper components in Jinlong Capsules.

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Claims (10)

1. a kind of method of quality control for detecting long-nosed pit viper ingredient in Jinlong capsule, it is characterised in that step is as follows：

(1) DNA in Jinlong capsule sample is extracted；

(2) Taqman probes are designed and synthesized；

(3) Taqman qPCR standard curves are established；

(4) Taqman qPCR detections are carried out.

2. according to the method described in claim 1, it is characterized in that, in the step (1), using the SDS extracting methods of improvement Carry out DNA extractions.

3. according to the method described in claim 2, it is characterized in that, the reagent for DNA extractions includes buffer solution A, buffer solution B With buffer solution C, wherein the buffer solution A include 1M Tris-HCL, 5M NaCL, 0.5M EDTA and lauryl sodium sulfate, institute Stating buffer solution B includes 3mol/L sodium acetates, and the buffer solution C includes 2mg/mL Proteinase Ks.

4. according to the method described in claim 3, it is characterized in that, the extraction operation of the step (1) is as follows：Take 2 gold dragons Capsule 's content adds in buffer solution A described in 2ml, buffer solution C described in 100 μ L, fully vibrates mixing, and 65 DEG C of water-bath 1.5h are added in Buffer solution B, 2mL component ratios are 24 described in 500 μ L：1 chloroform-isoamyl alcohol extracting, supernatant add in isometric isoamyl alcohol -20 DEG C precipitation 3 it is small when, 12000rpm centrifugation 20min, wash precipitation with 75% ethyl alcohol, dries, add in TE dissolve, purifying DNA.

5. according to the method described in claim 1, it is characterized in that, in the step (2), according to long-nosed pit viper specific primer design And Taqman probes are synthesized, 5 ' end fluorescent decorations of the Taqman probes have a reporter group, and 3 ' terminal modified have quenching group；Report Group is accused as one kind in FAM, HEX, TAMRA, ROX, CY5, one kind in quenching group Dabcyl, BHQ1, BHQ2.

6. the according to the method described in claim 5, it is characterized in that, nucleotide sequence of the Taqman probes such as SEQ ID Shown in NO.1.

7. the according to the method described in claim 5, it is characterized in that, nucleotide sequence such as SEQ of the long-nosed pit viper specific primer Shown in ID NO.2 and SEQ ID NO.3.

8. according to the method described in claim 1, it is characterized in that, the step (3) includes preparing standard items, wherein will be from begging The DNA that snake musculature extracts is expanded with Cytb sequences universal primer；Reaction system is：PCR Taq premixs enzyme 20 μ L, on Each 2 μ L of anti-sense primer and final concentration of 200nmol/L, 12 μ L of deionized water, 4 μ L of template, reaction total volume are 40 μ L；React item Part is：94℃5min；94 DEG C of 30s, 53 DEG C of 60s, 72 DEG C of 60s, totally 35 cycle；72℃7min；Purify long-nosed pit viper Cytb segments PCR Product and as standard items through sequencing identification, bioassay standard product DNA concentration and purity, standard items are carried out with deionized water 10 multiple proportions gradient dilutions using the standard items after dilution as template, carry out real-time fluorescence quantitative PCR and establish standard curve.

9. according to the method described in claim 1, it is characterized in that, in the step (4), reaction system is：2 μ L of DNA profiling, 0.2 μ L of probe, each 0.4 μ L of upstream and downstream primer and final concentration of 200nmol/L, Bester qPCR MasterMix Taqman10 50 × 0.2 μ L of μ L, ROX, 6.8 μ L of deionized water, reaction total volume are 20 μ L.

10. according to the method described in claim 1, it is characterized in that, in the step (4), PCR response procedures are：95 DEG C pre- It is denatured 2min；95 DEG C of denaturation 10s, 50 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 40 cycle, at the end of each cycle extension Gather fluorescence signal.