CN107881243B

CN107881243B - Fluorescent quantitative PCR detection method for authenticity identification of bezoar and application thereof

Info

Publication number: CN107881243B
Application number: CN201711041967.4A
Authority: CN
Inventors: 张文娟; 魏锋; 马双成
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2017-10-31
Filing date: 2017-10-31
Publication date: 2022-03-18
Anticipated expiration: 2037-10-31
Also published as: CN107881243A

Abstract

The invention relates to a probe primer and a detection method for fluorescence quantitative PCR detection for authenticity identification of bezoar, which can be used for authenticity identification of bezoar medicinal materials, finished preparations and other related products. The invention also provides application of the probe primer in fluorescent quantitative PCR detection for authenticity identification of bezoar, wherein the probe primer is SEQ ID NO:1-3, 4-6, 7-9, 10-12, 13-15 and/or 16-18. The method adopts a fluorescent quantitative PCR probe method to detect the required animal-derived components and the non-required animal-derived components in the sample. The method is applied to the quality control of calculus bovis medicinal materials, finished preparations and other related products, and has the advantages of strong specificity, high sensitivity and good repeatability.

Description

Fluorescent quantitative PCR detection method for authenticity identification of bezoar and application thereof

Technical Field

The invention belongs to the technical field of medicines, relates to a medicine identification and detection method, and particularly relates to a probe primer and a detection method for fluorescence quantitative PCR detection for authenticity identification of bezoar.

Background

Natural calculus bovis is a dry gallstone of cattle (Bos taurus domesticus Gmelin) of Bos family, mainly contains bilirubin, bile acid, cholesterol, inorganic elements, protein, amino acids and other ingredients, has the effects of clearing away heart-fire, inducing resuscitation, cooling liver, removing toxic substances, eliminating phlegm, calming endogenous wind and the like, and is one of rare Chinese medicinal materials [1-2 ]. Because of the shortage of resources and high price, the research on the substitute, namely cultivated bezoar, in-vitro cultivated bezoar and artificial bezoar begins in the middle of the 50 s of the 20 th century in China.

The cultivated bezoar is prepared by inserting yellow factor into gallbladder of cattle by surgical operation method using living cattle body. The in vitro cultivation of the bezoar is to cultivate the bovine bilirubin calcium calculus by applying the modern bioengineering technology according to the principle and biochemical process of in vivo formation of the bilirubin calcium calculus and simulating the principle and biochemical process of in vivo gallstone formation in the in vitro bovine gallbladder bile. The artificial bezoar is prepared from ox gall powder, cholic acid, hyodeoxycholic acid, taurine, bilirubin, cholesterol, trace elements, etc.

The artificial cultivated bezoar is formed under the same specific ecological factor condition as natural bezoar, and has no obvious difference from natural bezoar in physical and chemical properties, taste, color, effective component content, etc. The in vitro cultured calculus bovis is similar to natural calculus bovis in properties, structure, components and main component content. The difference between the artificial bezoar and the natural bezoar is large [3-7 ].

The bezoar has wide application in the preparation of the prescription, the resource is short, especially the natural bezoar and the cultivated bezoar have long production period and low yield, which belong to rare medicinal materials, and the situation of adulteration and counterfeiting caused by frequent exposition in the market recently. In order to prevent the occurrence of the phenomenon, a new detection means is urgently needed to be developed, supervision is enhanced, and the quality of the calculus bovis medicinal materials is better controlled.

Disclosure of Invention

The invention establishes a probe primer and a detection method for fluorescence quantitative PCR detection for authenticity identification of bezoar for the first time, and the probe primer and the detection method can be used for identification of bezoar medicinal materials and related products thereof.

In one aspect, the invention provides a fluorescent quantitative PCR detection method for authenticity identification of bezoar, which comprises the following steps:

weighing a bezoar sample and pretreating the sample;

extracting DNA from the sample and preparing a DNA template; and

selecting a probe primer;

optimizing reaction conditions;

amplifying the DNA template by using the probe primer;

according to the amplification result, judging a positive result according to a set CT value critical point;

the method can be used for detecting the bovine-derived components and the non-bovine-derived components in the sample, wherein the non-bovine-derived components comprise other animal-derived components such as pigs, horses, donkeys, sheep and the like.

In some embodiments, the probe primers are selected from one or more of the following groups: the sequence numbers of the probe primers are SEQ ID NO 1-3, SEQ ID NO 4-6, SEQ ID NO 7-9, SEQ ID NO 10-12, SEQ ID NO 13-15 and SEQ ID NO 16-18. Wherein each set of probe primers comprises three sequences, and wherein the three sequences comprised in any one set are used simultaneously without resolution when used for detection.

In other embodiments, the probe primers are probe primers of SEQ ID NO 13-15 and/or 16-18. In other embodiments, the probe primers are probe primers of SEQ ID NOs 1-3, 4-6, 7-9, and/or 10-12.

In one embodiment of the present invention, the sample is a bezoar bovis traditional Chinese medicine or a formulated preparation containing bezoar bovis.

In one embodiment of the present invention, the step of extracting DNA is quantitative and purity determination using uv spectrophotometry.

In one embodiment of the invention, the step of selecting probe primers is performed by selecting said probe primers to be cytb group probe primers for swine, CO I or ATP probe primers for horse, CO I probe primers for cattle, or LOOP or ND5 probe primers for donkey by looking at specificity and sensitivity.

In one embodiment of the invention, the reaction conditions include: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and fluorescence collection at 60 ℃.

In one embodiment of the present invention, when the probe primers are the probe primers of the cytb group, the set CT value critical point is 40, i.e., when the CT value is less than 40, a positive result is determined.

The detection method adopts a TaqMan probe method in fluorescent quantitative PCR. The main principle of the TaqMan probe method is that a fluorescent reporter group and a quenching group are respectively marked at two ends of a probe. When the primer probes are specifically combined with the DNA template, the probes are cut off by the exonuclease activity of the DNA polymerase, and fluorescent groups are dissociated out to detect fluorescent signals; when the probe or primer fails to bind to the template, the probe is in an intact state and the fluorescence of the reporter is absorbed by the quencher, so that no fluorescent signal is detected [8-11 ]. The inventor finds that the TaqMan probe method has better specificity compared with the common PCR method and the dye fluorescent quantitative PCR method by virtue of double functions of the probe and the primer, and has high sensitivity by virtue of a judgment result of the generation condition of a fluorescent signal. The research of the invention shows that based on the characteristics, the method is especially suitable for the sample with less target DNA content or possibly mixed multiple species, and has good application prospect in drug detection.

On the other hand, the invention provides application of a probe primer in fluorescent quantitative PCR detection for authenticity identification of bezoar, wherein the probe primer is SEQ ID NO:1-3, 4-6, 7-9, 10-12, 13-15, and/or 16-18, wherein each set of probe primers comprises three sequences, and wherein the three sequences comprised in any set are used simultaneously without resolution when used for detection.

The probe primer and the method can be used for detecting the calculus bovis traditional Chinese medicinal materials or the calculus bovis-containing formulated preparation. In addition, the probe primer and the method can also be used for detecting horse/cattle-derived components of meat, rubber medicinal materials and the like.

In yet another aspect, the invention provides self-designed probe primers consisting of SEQ ID NO:13-15 or 16-18.

The inventor finds that the calculus bovis medicinal material has low content of histiocytes and low abundance of DNA, is difficult to detect by using a common PCR method, and can easily detect DNA from various sources by using a TaqMan probe method. Except TaqMan probes, MGB probes can also be adopted, and the type of probes can distinguish the difference of 1 base and can be applied to detection of genotyping, virus variation and the like.

Drawings

FIG. 1 is a graph depicting the amplification of porcine DNA templates by two sets of probe primers, according to one embodiment of the present invention, wherein: a and B correspond to the cases of the cytb groups (SEQ ID NOS: 1-3) and ND2 groups, respectively.

FIG. 2 is an amplification curve (SEQ ID NOS: 1-3) of a medium positive sample (porcine-derived component-containing bovine bezoar artifacts) according to an embodiment of the present invention.

FIG. 3 is a graph depicting the amplification of other common closely related species by porcine cytb probe primers (SEQ ID NOS: 1-3) wherein A, B and C are curves for amplification of DNA from donkey, horse, and bovine sources, respectively.

FIG. 4 depicts the results of 3 replicates of the same sample (SEQ ID NOS: 1-3) according to one embodiment of the present invention.

FIGS. 5A-5B are the results of the detection of bovine skin and bovine bezoar samples using self-designed bovine probe primers (SEQ ID NOS: 16-18). A, B, C and D are curves for amplifying DNA from cow's hide, natural bezoar, cultured bezoar and artificial bezoar.

Detailed Description

The present invention is described in further detail below with reference to the drawings and examples, which are illustrative only and therefore should not be construed as limiting the scope of the present invention.

Instrument and reagent

Real-time fluorescent quantitative PCR instrument (AB 7500FAST), analytical balance (Mettler AB135-S), water purifier (Millipore Co.), ball mill (M400, Retsch), micro ultraviolet spectrophotometer, high speed centrifuge, metal heater. Blood/tissue/cell genome extraction kit (DP304, Tiangen Biotechnology Ltd.),

universal Master Mix II, probes and primers.

Second, sample collection

Collecting 3 batches of cultured bezoar counterfeit product Guangxi and 2 batches of Sichuan, and 1 batch of cultured bezoar genuine product, natural bezoar and artificial bezoar respectively (Table 1).

TABLE 1 sample information Table

Sample numbering	Sample name	Origin/place of origin
			PZNH_WP01	Cultivated bezoar medicine	Guangxi province
PZNH_WP02	Cultivated bezoar medicine	Guangxi province
			PZNH_WP03	Cultivated bezoar medicine	Sichuan
PZNH_WP04	Cultivated bezoar medicine	Sichuan
			PZNH-HZ	Cultivated bezoar medicine	Guangxi province
PZNH_SD	Cultivated bezoar genuine product	Provided by Shanxi province
			NH_SD	Natural bezoar	Chinese food and drug testing research institute control medicinal material
RGNH_SD	Artificial bezoar	Chinese food and drug testing research institute control medicinal material

Second, establishment of fluorescent quantitative PCR detection method

1. DNA extraction

Extracting by using a common kit for extracting animal tissues, and performing quantification and purity determination by adopting an ultraviolet spectrophotometry, wherein the inventor finds that about 20-100 ng of DNA can be obtained from about 10mg of a sample generally; these DNAs were dissolved in 50uL TE, 0.5 to 1uL was used as a template, and the CT value of the detection result was generally 30 or less or about 30. The purity OD260/280 of the DNA is often difficult to reach 1.7, but has little influence on the final detection result. The method is well-tolerated for the quantity and quality of template DNA.

2. Selection of Probe primers

Two sets of probe primers for swine (Sus scrofa bred) were found in the literature, located at cytochrome b (cytb) and NADH dehydrogenase outbunit 2(ND2), respectively. The cytb group probe primers can effectively amplify the DNA template of the pig, and present a typical S-shaped amplification curve, and the CT value is about 20. The ND2 group showed no typical sigmoidal curve, i.e., no efficient amplification of the porcine DNA template (fig. 1). Therefore, the method selects the cytb group probe primer for experiment. Specific probes for cattle, horses and donkeys were also screened.

According to one embodiment, the invention relates to a probe method for a fluorescent quantitative PCR method, comprising different fluorescent signals or non-fluorescent signal labels (e.g.FAM, TAMARA, BQ, etc.), different types of probes (e.g.TaqMAN probes, MGB probes, etc.) with the same probe/primer sequence.

In the selection of the probe/primer, the reported probe/primer is screened to find the optimal probe/primer suitable for the detection purpose (investigating specificity and sensitivity), and a novel probe/primer designed by self is also provided.

In some embodiments, the probe primers used in the present invention are shown in table 2.

TABLE 2 Probe primers

Note: (1) each group of probe primers comprises three sequences, and the probes are not separable when used simultaneously in an experiment; (2) the sequence 1-3 is used for detecting pig-derived components in a sample, the sequence 4-6 is used for detecting horse-derived components in the sample, the sequence 7-9 and the sequence 10-12 are used for detecting donkey-derived components in the sample, the sequence 13-15 is used for detecting horse-derived components in the sample, and the sequence 16-18 is used for detecting cattle-derived components in the sample; (3) the sequences 1-3, 4-6, 7-9 and 10-12 are introduced from the literature, and the sequences 13-15 and 16-18 are designed by the inventor; (4) the sequence information is also listed in the sequence listing attached to this application.

3. Selection of reaction conditions

The reaction condition adopts the conventional TaqMan probe method fluorescent quantitative PCR condition, namely, the pre-denaturation is carried out for 5min at the temperature of 94 ℃; denaturation at 94 ℃ for 30S, annealing at 60 ℃ for 45S, and fluorescence collection at 60 ℃ to obtain a more typical S-type amplification curve for the positive control group (FIG. 1). Amplification usually reaches a plateau at the latest of 46-48, so the fluorescence signal is collected for a total of 50 cycles (FIG. 2).

4. Determination of CT value critical point

Taking cultivated bezoar as an example, the CT value of the positive sample is usually less than 35, and no case of more than 40 is found (as shown in figure 2), so the CT value critical point of the positive result is positioned at 40, namely the CT value is less than 40, the positive result is judged, and the pig-derived components are detected. The positive results of other probes in the detection are determined by the same method to judge the CT value.

5. Species-specific investigation of Probe primers

The amplification of DNA from donkey, horse and cow with pig specific probe primers showed no typical sigmoid curve, i.e., all the results were negative (FIG. 3). The probe primer was found to have good specificity. The specificity of the other probes was also tested.

6. Examination of applicability

5 batches of cultivated bezoar counterfeit products and 1 batch of cultivated bezoar genuine products are detected by using the method. Pig-derived components were detected in all of the 5 counterfeit batches, and no pig-derived components were detected in the 1 authentic batch (Table 3). The method is good in applicability.

TABLE 3 test results of bezoar samples

7. Intermediate precision

Taking the cultivated calculus bovis medicinal material as an example, in the same experiment, two repeated experimental groups of the cultivated calculus bovis reference medicinal material have negative results; 5 batches of cultured bezoar counterfeit products have a difference (Δ CT) between CT values of two parallel test groups of 0.14-0.95 (Table 2).

8. Repeatability of

Taking cultivated calculus bovis as an example, the same positive sample (cultivated calculus bovis counterfeit product containing pig-derived components) is subjected to three times of experiments by using the method, and a consistent result is obtained, namely the sample is detected to contain the pig-derived components three times (figure 4). The method is seen to be highly reproducible.

9. Detection of bovine-derived components in bezoar medicinal materials

The method is characterized in that a self-designed bovine probe primer is used for detecting the bovine-derived component conditions in several different bovine-derived medicinal materials. As a result, bovine-derived components were successfully detected in all of natural bezoar, cultured bezoar and artificial bezoar. Bovine hide was selected as a positive control (fig. 5A-5B).

Third, summarize and discuss

Natural calculus bovis is gall bladder calculus formed by cattle under natural conditions, resources are limited, although cultivated calculus bovis is artificially cultivated and produced, the cultivated calculus bovis still belongs to precious and rare medicinal materials due to long production period, low yield and large market demand, the phenomenon of adulteration and counterfeiting is serious, particularly, counterfeit products containing pig-derived components are more common, and the intensive supervision and supervision of the counterfeit products are urgently needed.

The authenticity identification is most accurate from the aspect of DNA judgment, and the DNA molecule identification method based on the TaqMAN probe, which is applied by the method, greatly improves the specificity compared with the common PCR method due to the double identification of the probe and the primer, and improves the sensitivity of the method to a great extent by the detection means based on the fluorescent signal. The method has strong specificity, high sensitivity and good repeatability, and has no precedent application in the traditional Chinese medicine detection so far, so the establishment of the method is urgent and necessary.

The content of histiocyte in the bezoar medicine sample is less, and the acquisition amount of DNA is less. The method has high sensitivity, is suitable for testing medicinal materials such as cultured bezoar, and is advantageous, however, experimenters are required to be trained by regular molecular biology testing skills, the experimental operation is correct, and in the operation process, the external pollution interference is prevented. The method has good repeatability, and can better avoid the situation of false negative or false positive generally as long as the operation is strictly standardized. The method is applied to actual inspection work, and can seriously attack the phenomenon of adulteration of the calculus bovis medicinal materials in the market, thereby providing powerful technical support for quality control and market supervision of the calculus bovis medicinal materials.

Specific embodiments are described in detail herein, however, this is by way of example for purposes of illustration only and is not intended to limit the scope of the claims which follow. It should be understood that various substitutions, alterations and modifications to the embodiments described herein may be made without departing from the spirit and scope of the invention as defined by the appended claims and shall therefore fall within the scope of the invention as hereinafter claimed.

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Claims

1. A fluorescence quantitative PCR detection method for identifying the authenticity of bezoar comprises the following steps:

weighing a bezoar sample and pretreating the sample;

extracting DNA from the sample by adopting a kit for extracting animal tissue DNA and preparing a DNA template;

selecting a probe primer;

optimizing reaction conditions, including: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and collecting fluorescence at 60 ℃;

amplifying the DNA template with the probe primer, wherein amplification is for 50 cycles;

wherein the probe primer comprises: the primer probe for pigs is shown as SEQ ID NO. 1-3, the primer probe for horses is shown as SEQ ID NO. 4-6 or SEQ ID NO. 13-15, the primer probe for donkeys is shown as SEQ ID NO. 7-9 or SEQ ID NO. 10-12 and the primer probe for cattle is shown as SEQ ID NO. 16-18;

wherein, the detection method adopts a TaqMan probe method in fluorescent quantitative PCR.

2. The method of claim 1, wherein the sample is a bovine bezoar Chinese medicinal material or a formulated preparation containing bovine bezoar.

3. The method of claim 1, wherein said step of extracting DNA is quantitative and purity determination using ultraviolet spectrophotometry.