CN103937881B - Differentiate fluorescence PCR detection reagent and the preparation method and application in cow genome source in heparin - Google Patents

Abstract

The invention discloses a fluorescent PCR detection reagent for identifying the origin of bovine genes in heparin, a preparation method and an application thereof. Through sequencing and comparison of bovine genes, a detection method for detecting the origin of bovine components in crude heparin products is provided, real-time Fluorescent quantitative PCR primers and probes, the length of the amplified target fragment for detecting bovine components is 92bp, and the invention also provides a kit for quantitatively detecting bovine genes. The detection method and the detection kit of the invention have the advantages of accurate detection, high sensitivity, strong specificity, etc., and have good specimen detection ability.

Description

Fluorescent PCR detection reagent for identifying bovine gene source in heparin, preparation method and application

technical field

The invention relates to a biological detection reagent capable of identifying the source of bovine genes in heparin, as well as a preparation method and application of the reagent.

Background technique

Heparin is an anticoagulant, which is a polymer composed of two polysaccharides connected alternately, and has anticoagulant effects both in vivo and in vitro. Clinically, it is mainly used for thromboembolic diseases, myocardial infarction, cardiovascular surgery, cardiac catheterization, extracorporeal circulation, hemodialysis, etc. With the progress of pharmacology and clinical medicine, the application of heparin has been expanding.

In 2010, the export volume of my country's heparin and its salt hit a record high, and it has become my country's largest export commodity of western medicine. In 2010, my country's heparin and its salts were exported to 51 countries and regions, with a high degree of export concentration. The top five export markets were France, the United States, Germany, Austria and Italy, accounting for a cumulative 86.32% of exports. Heparin raw materials can be directly used to make standard heparin preparations, or further processed to make low molecular weight heparin raw materials and then made into low molecular weight heparin preparations. Standard heparin preparations and low molecular weight heparin preparations can be directly applied to clinical treatment. Heparin products mainly include crude heparin, heparin raw materials, standard heparin preparations, low-molecular-weight heparin raw materials and low-molecular-weight heparin preparations.

Since heparin drugs are mainly used in the treatment of cardiovascular and cerebrovascular diseases and hemodialysis, among them, they are the only effective specific drugs in the treatment of severe hemodialysis, and their users are concentrated in the elderly and obese people, and the main consumer markets are concentrated in Europe and the United States and developed countries such as Japan. The demand for heparin raw materials in the international market is very strong, mainly due to the rapid expansion of the market for its downstream products, heparin drugs, and maintain a high-speed growth trend. It is estimated that by 2015, the global market size of heparin drugs will reach 7.9 billion US dollars, which means that the demand for heparin in the global pharmaceutical market will still grow strongly. However, after the "heparin sodium incident" in 2008, global heparin drug companies paid close attention to the quality of heparin sodium raw materials, and the demand for heparin sodium raw materials certified by FDA and CEP increased significantly, while those who did not have this quality certification Enterprises will not be able to gain a foothold in the international market in the future. Therefore, the quality inspection of heparin sodium has become a hot issue, and the new version of the Pharmacopoeia has made clear regulations on the source inspection of heparin sodium for the following reasons:

Heparin is first named after its discovery in the liver. It also exists in the lungs, blood vessel walls, intestinal mucosa and other tissues. It is a natural anticoagulant substance in animals. Naturally present in mast cells, it is now mainly extracted from bovine lung or pig small intestinal mucosa. However, because ruminants such as cattle may carry pathogenic prion proteins, they may suffer from transmissible spongiform encephalopathy (commonly known as sheep scrapie and mad cow disease). Humans may be infected with CJD by animals carrying the virus. Due to bovine spongiform encephalopathy and other viral diseases in animals, the uniqueness of the porcine source is a key requirement to ensure the safety of heparin. Therefore, in order to ensure the safety of the heparin supply chain, countries clearly require that heparin APIs are not allowed to come from cattle in terms of heparin imports. Therefore, it is particularly necessary to develop an efficient reagent and method for detecting the source of heparin.

At present, the detection methods of harmful impurities in crude heparin mainly include nuclear magnetic resonance, high performance liquid chromatography and other methods. With the rapid development of molecular biology technology, PCR technology has been developed rapidly. Since heparin needs to be extracted and purified many times during the preparation process, the DNA in it will be affected and degraded into many small fragments. If the target fragment is too large, positive fragments may not be found in the test. However, limited by the effectiveness of electrophoretic detection, it is difficult to adopt small fragment target products in general PCR. Chinese Patent Application No. 200810123709.5 disclosed by Huang Yahong, Hou Yayi et al. a nested PCR method to identify the source of animal genes in heparin. However, the general PCR method requires gel electrophoresis of the PCR product to complete the entire detection, and the detection sensitivity of the electrophoresis band is low. When the gene concentration difference in the sample is not large, it is difficult to reflect the difference in the brightness of the band, which in turn affects the detection of the difference. Determination of the presence or absence of bovine genes in heparin. Moreover, gel electrophoresis detection will prolong the entire detection time and delay the detection progress. Therefore, this method has problems such as low detection sensitivity and complicated operation. Chinese Patent Application No. 200910094768.9 discloses a fluorescent PCR detection reagent for identifying ruminant-derived components and its preparation method and application. It involves a biological detection reagent that can simultaneously identify three ruminant species including cattle, goats and sheep. , and the preparation method and application of this reagent. However, this system is not suitable for the detection of bovine genes in heparin. The main reasons can be summarized as the following three points: 1) The research results of many people show that heparin polysaccharides have an inhibitory effect on PCR amplification enzymes and cannot be directly used in real- The time PCR experiment must be processed to be feasible; 2) The method in this patent is relatively complicated, and multiplex PCR is not conducive to the detection of bovine genes in heparin; 3) In addition, the reaction system of this method is 50 μl, which consumes a lot of reagents , which greatly increases the detection cost, which is not conducive to the realization of large-scale detection in enterprises.

Contents of the invention

The invention provides a fluorescent quantitative PCR detection reagent capable of identifying and detecting the source composition (bovine gene) of crude heparin samples, as well as its preparation method and application. The present invention uses the TaqMan fluorescence quantitative PCR method to overcome the shortcomings of ordinary PCR, and is the best method for detecting ruminant genes in heparin raw materials, and has the advantages of high sensitivity, wide linear range, short analysis time, simple operation and good repeatability , The results are intuitive and so on.

The purpose of the present invention is achieved through the following technical solutions:

A biological detection reagent for identifying the source of bovine genes in heparin, comprising a pair of primers and a TaqMan probe, the pair of primers are respectively the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the Described TaqMan probe is the nucleotide sequence shown in SEQ ID NO.3.

Further, the 5' end of the TaqMan probe SEQ ID NO.3 is connected with a fluorescent reporter group, and the 3' end is connected with a quenching group.

Further, the fluorescent reporter group connected to the 5' end is 6-carboxyfluorescein (FAM), and the quencher group connected to the 3' end is 6-carboxytetramethylnordamine (TAMRA). The full name of the above-mentioned FAM is 6-carboxy-fluorescein, which is 6-carboxyfluorescein labeled at the 5' end of the probe; the full name of TAMRA is Carboxytetramethylrhodamine, which is 6-carboxytetramethylnordamine labeled at the 3-end of the probe.

Another object of the present invention is achieved through the following technical solutions:

A preparation method of a biological detection reagent for identifying bovine gene sources in heparin, the preparation method comprising the following steps:

1) Select the specific and conserved conserved sequence fragment of the Bov-A2 gene in the bovine mitochondrial gene as the target, and the amplified target nucleotide sequence of the gene fragment is SEQ ID NO.4;

2) According to the characteristics of cow-specific and conserved mitochondrial gene conserved sequences, use Primer Express3.0 software and PrimerSelect software in DNAStar to design and synthesize primers and probes to be tested;

3) Synthesis of primers and probes OligoDNA was synthesized using a fully automatic DNA synthesizer;

4) The probe is synthesized and labeled at both ends at the same time. The fluorescent reporter group labeled at the 5' end of the probe is FAM (6-carboxyfluorescein), and the group labeled at the 3' end is TAMRA (6-carboxytetramethylnodan bright);

5) After the designed and synthesized primers and probes were subjected to the optimal pairing screening experiment, the above-mentioned primers and probes were obtained.

The application of the biological detection reagent for identifying the origin of bovine gene in heparin, the biological detection reagent for identifying the origin of bovine gene in heparin can be used to prepare a fluorescent quantitative PCR standardization kit.

Another object of the present invention is achieved through the following technical solutions:

A biological detection method for identifying the source of bovine genes in heparin, the detection method comprising the following steps:

A. Pretreatment of heparin to be tested

(1) Add 0.03g of powdered heparin sample to a sterilized centrifuge tube, add 1ml of enzyme-free water to the tube, shake and mix well, centrifuge at 3500rpm, 30s, 100℃ water bath for 5min, let cool to room temperature;

(2) Repeatedly draw and stir the tip of the pipette until the sample is dissolved. Take 10 μl of the dissolved sample solution into a new sterilized centrifuge tube, then add 990 μl of enzyme-free water, shake and mix well, and use a 1ml syringe to pass the solution through a 45 μm micropore Membrane filtration;

(3) Take 45 μl of the solution obtained in the previous step, add 0.7 μl of reagent heparinase, shake and mix, and bathe in water at 28°C for 18 hours to obtain the heparin sample to be tested;

B. Fluorescent quantitative PCR

(1) PCR reaction system

Fluorescent quantitative PCR uses a 25 μl volume of reaction solution, and the reaction amplification system and amplification conditions are carried out according to the following reaction parameters:

The total volume of the real-time fluorescent quantitative PCR reaction is 25 μl, and the following components are added to the reaction tube, and the reaction system is:

MIX 12.5μl

upstream primer F 0.5μl downstream primer R 0.5μl probe 6.5μl Heparin samples to be tested 5μl

The upstream primer F and the downstream primer R are respectively the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe is the nucleotide sequence shown in SEQ ID NO.3;

(2) PCR amplification conditions

The amplification conditions of real-time fluorescence quantitative PCR are:

C. Result Analysis and Judgment

(1) Result analysis condition setting

According to the fluorescein type of the amplification curve, FAM represents the bovine-derived component, and the test result can be read directly; the baseline and threshold setting principles are adjusted according to the instrument, and the threshold line just exceeds the highest point of the normal negative control amplification curve;

(2) Negative and positive controls are set up during sample testing. When the two controls are effectively amplified in the test, the results are judged according to the following criteria:

A sample with a Ct value greater than 40 is a negative result: a Ct value greater than 38 or no amplification curve indicates that there is no bovine-derived component in the sample;

Samples with a Ct value less than or equal to 35 are positive results: the Ct value is less than or equal to 35, and an obvious amplification curve appears, indicating the presence of bovine-derived components in the sample.

The beneficial effect of the present invention compared with prior art is:

1. The present invention can determine the source of animal genes contained in crude heparin and raw materials according to requirements, and both crude heparin and refined heparin are applicable;

2. The TaqMan fluorescent quantitative PCR method used in the present invention overcomes the shortcomings of common PCR, and is the best method for detecting ruminant genes in heparin raw materials, with high sensitivity, wide linear range, short analysis time, easy operation, Good repeatability and intuitive results;

3. The method of the present invention for the accurate detection of low-content bovine-derived components in samples or the degradation of bovine-derived component DNA into small fragments after high-temperature and high-pressure treatment has high sensitivity, high specificity and high accuracy;

4. The bovine gene standard curve of the method of the present invention reaches R2≥0.99 after many tests; gene detection range: 0.001～100ng/ml; gene detection limit: >0.01ng/ml;

5. The present invention establishes a specific, sensitive, safe, accurate and rapid detection method for bovine-derived heparin, which is of great significance in many aspects such as effective supervision and strict elimination of false phenomena caused by adulteration of cattle products.

Description of drawings

Figure 1 is a real-time fluorescence quantitative PCR detection bovine gene standard curve, where the abscissa is the logarithmic value of the positive template concentration, and the ordinate is the Ct value of the reaction system to detect different concentrations of positive templates. The template concentration marked by numbers in the figure is 1:100ng /ml; 2: 10ng/ml; 3: 1ng/ml; 4: 0.1ng/ml; 5: 0.01ng/ml; 6: 0.001ng/ml, R2=0.993;

Fig. 2 is a diagram showing the results of detection of bovine genes in crude heparin by the present invention: 1 indicates that there are bovine-derived components in the sample, and 2 indicates that there is no bovine-derived component in the sample;

Fig. 3 is a diagram showing the results of the detection of bovine genes in fine-quality heparin according to the present invention: 1 indicates that there are bovine-derived components in the sample, and 2 indicates that there is no bovine-derived component in the sample.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting 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 technical means used in the embodiments are conventional means well known to those skilled in the art.

Example 1:

A biological detection reagent for identifying the source of bovine genes in heparin, comprising a pair of primers and a TaqMan probe, the pair of primers are respectively the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the Described TaqMan probe is the nucleotide sequence shown in SEQ ID NO.3.

Further, the 5' end of the TaqMan probe SEQ ID NO.3 is connected with a fluorescent reporter group, and the 3' end is connected with a quenching group.

Further, the fluorescent reporter group connected to the 5' end is 6-carboxyfluorescein (FAM), and the quencher group connected to the 3' end is 6-carboxytetramethylnordamine (TAMRA).

A preparation method of a biological detection reagent for identifying bovine gene sources in heparin, the preparation method comprising the following steps:

1) Select the specific and conserved conserved sequence fragment of the Bov-A2 gene in the bovine mitochondrial gene as the target, and the amplified target nucleotide sequence of the gene fragment is SEQ ID NO.4;

2) According to the characteristics of cow-specific and conserved mitochondrial gene conserved sequences, use Primer Express3.0 software and PrimerSelect software in DNAStar to design and synthesize primers and probes to be tested;

3) Synthesis of primers and probes OligoDNA was synthesized using a fully automatic DNA synthesizer;

4) The probe is synthesized and labeled at both ends at the same time. The fluorescent reporter group labeled at the 5' end of the probe is FAM (6-carboxyfluorescein), and the group labeled at the 3' end is TAMRA (6-carboxytetramethylnodan bright);

5) After the designed and synthesized primers and probes were subjected to the optimal pairing screening experiment, the above-mentioned primers and probes were obtained.

The application of the biological detection reagent for identifying the origin of bovine gene in heparin, the biological detection reagent for identifying the origin of bovine gene in heparin can be used to prepare a fluorescent quantitative PCR standardization kit.

A biological detection method for identifying the source of bovine genes in heparin, the detection method comprising the following steps:

A. Pretreatment of heparin to be tested

(1) Add 0.03g of powdered heparin sample to a sterilized centrifuge tube, add 1ml of enzyme-free water to the tube, shake and mix well, centrifuge at 3500rpm, 30s, 100℃ water bath for 5min, let cool to room temperature;

(2) Repeatedly draw and stir the tip of the pipette until the sample is dissolved. Take 10 μl of the dissolved sample solution into a new sterilized centrifuge tube, then add 990 μl of enzyme-free water, shake and mix well, and use a 1ml syringe to pass the solution through a 45 μm micropore Membrane filtration;

(3) Take 45 μl of the solution obtained in the previous step, add 0.7 μl of reagent heparinase, shake and mix, and bathe in water at 28°C for 18 hours to obtain the heparin sample to be tested;

B. Fluorescent quantitative PCR

(1) PCR reaction system

Fluorescent quantitative PCR uses a 25 μl volume of reaction solution, and the reaction amplification system and amplification conditions are carried out according to the following reaction parameters:

The total volume of the real-time fluorescent quantitative PCR reaction is 25 μl, and the following components are added to the reaction tube, and the reaction system is:

MIX 12.5μl upstream primer F 0.5μl downstream primer R 0.5μl probe 6.5μl Heparin samples to be tested 5μl

The upstream primer F and the downstream primer R are respectively the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe is the nucleotide sequence shown in SEQ ID NO.3;

(2) PCR amplification conditions

The amplification conditions of real-time fluorescence quantitative PCR are:

C. Result Analysis and Judgment

(1) Result analysis condition setting

According to the fluorescein type of the amplification curve, FAM represents the bovine-derived component, and the test result can be read directly; the baseline and threshold setting principles are adjusted according to the instrument, and the threshold line just exceeds the highest point of the normal negative control amplification curve;

(2) Negative and positive controls are set up during sample testing. When the two controls are effectively amplified in the test, the results are judged according to the following criteria:

A sample with a Ct value greater than 40 is a negative result: a Ct value greater than 38 or no amplification curve indicates that there is no bovine-derived component in the sample;

Samples with a Ct value less than or equal to 35 are positive results: the Ct value is less than or equal to 35, and an obvious amplification curve appears, indicating the presence of bovine-derived components in the sample.

Example 2:

This embodiment is a preferred solution of embodiment 1, which is the detection of bovine genes in crude heparin.

1. Design primers and probes

In this example, the specific and conserved conserved sequence fragment of the Bov-A2 gene in the bovine mitochondrial gene is selected as the target, and by collecting a large number of bovine mitochondrial gene sequences reported in GenBank and the mitochondrial gene sequences of various other ruminants and non-ruminants, For sequence analysis and comparison, use PrimerExpress3.0 software and PrimerSelect software in DNAStar to design and synthesize primers and probes to be tested. After conducting the optimal pairing screening experiment on the designed and synthesized primers and probes, the candidate primers and probes are initially determined, after a large number of reaction conditions optimization, comparative tests and verification tests, and after a large number of sample detection application evaluations, the amplification efficiency is determined The primers and probes with the best specificity are designed based on the conserved sequence fragments of the specific and conserved cytb gene in the bovine mitochondrial gene. The length of the amplified target fragment is 92bp. The synthesis of the selected primers and probes is entrusted to Invitrogen Company , OligoDNA was synthesized using a fully automatic DNA synthesizer. The probe is synthesized and labeled at both ends at the same time. The fluorescent reporter group labeled at the 5' end of the probe is FAM, and the group labeled at the 3' end is TAMRA.

2. DNA extraction

The fresh beef was ground and crushed, and the total DNA was extracted with a DNA extraction kit. The OD260/OD280 value of the extracted DNA sample measured by the nucleic acid protein analyzer is not less than 1.5, and after electrophoresis detection, the DNA sample concentration is diluted to 100ng/ml according to the measured concentration, and stored at -20°C. When analyzing the sensitivity of primer amplification, the DNA was diluted 10 times (10 ² ～10 ^-3 ng/ml), and then the diluted DNA was used as a template for PCR amplification.

3. Pretreatment of crude heparin

(1) The crude heparin is in the form of fine particles. Grind the crude heparin into powder, weigh 0.03g of the powder sample into a sterilized centrifuge tube, add 1ml of enzyme-free water to the tube, shake and mix well, and centrifuge at 3500rpm for 30s. 100°C water bath for 5 minutes, let cool to room temperature;

(2) Repeatedly draw and stir the tip of the pipette until the sample is dissolved. Take 10 μl of the dissolved sample solution into a new sterilized centrifuge tube, then add 990 μl of enzyme-free water, shake and mix well, and use a 1ml syringe to pass the solution through a 45 μm micropore membrane filtration;

(3) Take 45 μl of the solution from the previous step, add 0.7 μl of reagent heparinase, shake and mix well, and bathe in water at 28°C for 18 hours to obtain the heparin sample to be tested.

4. Fluorescence quantitative PCR

(1) Kit components:

Enzyme-free water 20ml

PCR reagent 1.27ml Bovine DNA Primer 30μl bovine DNA probe 350μl Bovine DNA Standard Solution 36μl

(2) PCR reaction system

Fluorescent quantitative PCR uses 25 μl volume of reaction solution, using Bio-Rad MyiQ2 real-time fluorescent quantitative PCR instrument. The reaction amplification system and amplification conditions are carried out according to the following reaction parameters:

The total volume of the real-time fluorescent quantitative PCR reaction is 25 μl, and the following components are added to the reaction tube, and the reaction system is:

MIX 12.5μl upstream primer F 0.5μl downstream primer R 0.5μl probe 6.5μl Bovine DNA standard solution or heparin product to be tested 5μl

use

(3) PCR amplification conditions

The optimal reaction conditions for PCR were explored with the optimized reaction system, and verified through repeated experiments. Each experiment used the same amount of template in the same tube, and each reaction condition was repeated three times. The amplification conditions for real-time fluorescent quantitative PCR were finally determined as :

A control was set up, and two groups were set up for positive and negative controls.

4. Fluorescent quantitative PCR stability and repeatability experiments

When evaluating the reproducibility and stability of the real-time quantitative fluorescent PCR test of bovine-derived components, the same amount of DNA samples mixed in equal proportions of cattle are used each time, and the real-time quantitative fluorescent PCR detection is repeated under the same reaction conditions, with a setting of 6 Each experiment was repeated 12 times.

5. Drawing of standard curve

The concentration of the standard plasmid template was diluted to 1.0×10 ² ~1.0×10 ^-3 ng/ml, a total of 6 dilutions were used as standard templates for real-time quantitative fluorescent PCR. Taking the DNA concentration as the X-axis and the cycle number Ct as the Y-axis, a standard curve was obtained, as shown in Figure 1.

6. Result analysis and judgment

(1) Result analysis condition setting

According to the fluorescein type of the amplification curve, FAM represents the bovine-derived component, and the detection result is directly read. The baseline and threshold setting principles are adjusted according to the instrument, and the threshold line just exceeds the highest point of the normal negative control amplification curve.

(2) Negative and positive controls were set up during sample testing. When the two controls in the test are effectively amplified, the result judgment criteria are as follows:

A sample with a Ct value greater than 40 is a negative result: a Ct value greater than 38 or no amplification curve indicates that there is no bovine-derived component in the sample;

A sample with a Ct value less than or equal to 35 is a positive result: a Ct value less than or equal to 35, and an obvious amplification curve appears, indicating the presence of bovine-derived components in the sample;

According to the detection and screening of a large number of samples, it is determined that the Ct value is between 35 and 40 as suspicious samples, which need to be tested again.

Example 3:

This embodiment is the preferred solution of embodiment 1, which is the detection of bovine gene in high-quality heparin.

1. Design primers and probes

In this example, the specific and conserved conserved sequence fragment of the Bov-A2 gene in the bovine mitochondrial gene is selected as the target, and by collecting a large number of bovine mitochondrial gene sequences reported in GenBank and the mitochondrial gene sequences of various other ruminants and non-ruminants, For sequence analysis and comparison, primers and probes to be tested were designed and synthesized using Primer Express3.0 software and PrimerSelect software in DNAStar. After conducting the optimal pairing screening experiment on the designed and synthesized primers and probes, the candidate primers and probes are initially determined, after a large number of reaction conditions optimization, comparative tests and verification tests, and after a large number of sample detection application evaluations, the amplification efficiency is determined The primers and probes with the best specificity are designed based on the conserved sequence fragments of the specific and conserved cytb gene in the bovine mitochondrial gene. The length of the amplified target fragment is 92bp. The synthesis of the selected primers and probes is entrusted to Invitrogen Company Carry out the synthesis of OligoDNA using a fully automatic DNA synthesizer. The probe is synthesized and labeled at both ends at the same time. The fluorescent reporter group labeled at the 5' end of the probe is FAM (6-carboxyfluorescein), and the group labeled at the 3' end is TAMRA (6-carboxytetramethylnordamine). ;

2. DNA extraction

The fresh beef was ground and crushed, and the total DNA was extracted with a DNA extraction kit. The OD260/OD280 value of the extracted DNA sample measured by the nucleic acid protein analyzer is not less than 1.5, and after electrophoresis detection, the DNA sample concentration is diluted to 100ng/ml according to the measured concentration, and stored at -20°C. When analyzing the sensitivity of primer amplification, the DNA was diluted 10 times (10 ² ～10 ^-3 ng/ml), and then the diluted DNA was used as a template for PCR amplification.

3. High-quality heparin pretreatment

(1) Fine heparin is in the form of fine powder, so directly weigh 0.03g of fine heparin sample into a sterilized centrifuge tube, add 1ml of enzyme-free water to the tube, oscillate and mix, centrifuge at 3500rpm, 30s, 100℃ water bath for 5min, Let cool to room temperature;

(2) Repeatedly draw and stir the tip of the pipette until the sample is dissolved. Take 10 μl of the dissolved sample solution into a new sterilized centrifuge tube, then add 990 μl of enzyme-free water, shake and mix well, and use a 1ml syringe to pass the solution through a 45um micropore Membrane filtration;

(3) Take 45 μl of the solution from the previous step, add 0.7 μl of reagent heparinase, shake and mix well, and bathe in 28°C water for 18 hours.

4. Fluorescence quantitative PCR

(1) Kit components:

Enzyme-free water 20ml PCR reagent 1.27ml Bovine DNA Primer 30μl bovine DNA probe 350μl Bovine DNA Standard Solution 36μl

(2) PCR reaction system

Fluorescent quantitative PCR uses 25 μl volume of reaction solution, using Bio-Rad MyiQ2 real-time fluorescent quantitative PCR instrument. The reaction amplification system and amplification conditions are carried out according to the following reaction parameters:

The total volume of the real-time fluorescent quantitative PCR reaction is 25 μl, and the following components are added to the reaction tube, and the reaction system is:

MIX 12.5μl upstream primer F 0.5μl downstream primer R 0.5μl probe 6.5μl Bovine DNA standard solution or heparin product to be tested 5μl

(3) PCR amplification conditions

The optimal reaction conditions for PCR were explored with the optimized reaction system, and verified through repeated experiments. Each experiment used the same amount of template in the same tube, and each reaction condition was repeated three times. The amplification conditions for real-time fluorescent quantitative PCR were finally determined as :

A control was set up, and two groups were set up for positive and negative controls.

4. Fluorescent quantitative PCR stability and repeatability experiments

When evaluating the reproducibility and stability of the real-time quantitative fluorescent PCR test of bovine-derived components, the same amount of DNA samples mixed in equal proportions of cattle are used each time, and the real-time quantitative fluorescent PCR detection is repeated under the same reaction conditions, with a setting of 6 Each experiment was repeated 12 times.

5. Drawing of standard curve

The concentration of the standard plasmid template was diluted to 1.0×10 ² ~1.0×10 ^-3 ng/ml, a total of 6 dilutions were used as standard templates for real-time quantitative fluorescent PCR. Taking the DNA concentration as the X-axis and the cycle number Ct as the Y-axis, a standard curve was obtained, as shown in Figure 1.

6. Result analysis and judgment

(1) Result analysis condition setting

According to the fluorescein type of the amplification curve, FAM represents the bovine-derived component, and the detection result is directly read. The baseline and threshold setting principles are adjusted according to the instrument, and the threshold line just exceeds the highest point of the normal negative control amplification curve.

(2) Negative and positive controls were set up during sample testing. When the two controls in the test are effectively amplified, the result judgment criteria are as follows:

A sample with a Ct value greater than 40 is a negative result: a Ct value greater than 38 or no amplification curve indicates that there is no bovine-derived component in the sample;

A sample with a Ct value less than or equal to 35 is a positive result: a Ct value less than or equal to 35, and an obvious amplification curve appears, indicating the presence of bovine-derived components in the sample;

According to the detection and screening of a large number of samples, it is determined that the Ct value is between 35 and 40 as suspicious samples, which need to be tested again.

Therefore, the detection of bovine-derived components by fluorescent quantitative PCR has high sensitivity, strong specificity and good repeatability.

Claims (5)

1. A biological detection reagent for distinguishing bovine gene origin in heparin, comprising a pair of primers and a TaqMan probe, characterized in that, said pair of primers are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively Nucleotide sequence, the TaqMan probe is the nucleotide sequence shown in SEQ ID NO.3; The 5' end of the TaqMan probe SEQ ID NO.3 is connected with a fluorescent reporter group, and the 3' end is connected with Quenching group. 2. according to the biological detection reagent of bovine gene origin in the discrimination heparin described in claim 1, it is characterized in that, the fluorescent reporter group that described 5' end is connected is 6-carboxyfluorescein, and the described 3' end is connected The quenching group is 6‐carboxytetramethylnordamine. 3. a kind of preparation method of the biological detection reagent of bovine gene origin in the discrimination heparin as claimed in claim 1, described preparation method comprises the following steps: 1) Select the conserved sequence fragment of the specific and conserved Bov-A2 gene in the bovine mitochondrial gene as the target, and the target nucleotide sequence for amplification of the gene fragment is SEQ ID NO.4; 2) According to the characteristics of the cow-specific and conserved mitochondrial gene conservative sequence, use the Primer Express 3.0 software and the PrimerSelect software in DNAStar to design and synthesize primers and probes to be tested; 3) The synthesis of primers and probes uses a fully automatic DNA synthesizer to synthesize OligoDNA; 4) The probe is synthesized and labeled at both ends at the same time. The fluorescent reporter group labeled at the 5' end of the probe is FAM (6‐carboxyfluorescein), and the quencher group labeled at the 3' end is TAMRA (6‐carboxytetramethyl Nordamine); 5) After the designed and synthesized primers and probes are subjected to an optimal pairing screening experiment, the above-mentioned primers and probes are obtained. 4. The application of a biological detection reagent for identifying bovine gene source in heparin as claimed in claim 1 in the preparation of fluorescent quantitative PCR standardization kit. 5. A biological detection method for distinguishing bovine gene source in heparin, characterized in that, the detection method comprises the following steps: A. Pretreatment of heparin to be tested (1) Add 0.03 g of powdered heparin sample to a sterilized centrifuge tube, add 1 ml of enzyme-free water to the tube, oscillate and mix well, centrifuge at 3500 rpm for 30 s, and put in a water bath at 100 ° C for 5 min, then let cool to room temperature; (2) Repeatedly absorb and stir the tip of the pipette until the sample is dissolved, take 10 μl of the dissolved sample solution into a new sterilized centrifuge tube, then add 990 μl of enzyme-free water, shake and mix well, and use a 1ml syringe to pass the solution through a 45 μm micropore Membrane filtration; (3) Take 45 μl solution from the solution obtained in the previous step, add 0.7 μl reagent heparinase, shake to mix homogeneously, in a 28°C water bath for 18 hours, to obtain the heparin sample to be tested; B. Fluorescent quantitative PCR (1) PCR reaction system Fluorescent quantitative PCR uses a 25 μl volume of reaction solution, and the reaction amplification system and amplification conditions are carried out according to the following reaction parameters: The total volume of the real-time fluorescent quantitative PCR reaction is 25 μl, and the following components are added to the reaction tube, and the reaction system is: MIX 12.5μl upstream primer F 0.5μl downstream primer R 0.5μl probe 6.5μl Heparin samples to be tested 5μl The upstream primer F and the downstream primer R are respectively the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe is the nucleotide sequence shown in SEQ ID NO.3; (2) PCR amplification conditions The amplification conditions of real-time fluorescent quantitative PCR are: C. Result Analysis and Judgment (1) Result analysis condition setting According to the fluorescein type of the amplification curve, FAM represents the bovine-derived component, and the test result can be read directly; the baseline and threshold setting principles are adjusted according to the instrument, and the threshold line just exceeds the highest point of the normal negative control amplification curve; (2) Negative and positive controls are set up during sample testing. When the two controls are effectively amplified in the test, the results are judged according to the following criteria: A sample with a Ct value greater than 40 is a negative result: a Ct value greater than 38 or no amplification curve indicates that there is no bovine-derived component in the sample; A sample with a Ct value less than or equal to 35 is a positive result: the Ct value is less than or equal to 35, and there is an obvious Amplification curve, indicating the presence of bovine components in the sample. the