Primer probe combination for identifying antelope horn and product thereof

Abstract

The invention discloses a primer probe combination for identifying antelope horn and products thereof, and the primer probe combination comprises a forward primer, a reverse primer and a probe, and can be used for identifying the antelope horn and the products thereof. According to the TaqMan probe real-time fluorescent quantitative PCR method established by the invention, efficient amplification of a target fragment can be realized in a short time, the whole reaction is simple and rapid, the sensitivity is high, the repeatability is good, the stability is strong, and the application and popularization range is wide. The method disclosed by the invention can be applied to rapid identification of related detected materials in cases related to antelope horns and products thereof, and is particularly suitable for being used under the condition that the sample size is relatively large.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular identification, and in particular relates to a primer-probe combination for identifying saiga antelope horns and products thereof. Background Art

[0002] The saiga tatarica is a species of the genus Saiga in the family Bovidae, also known as the Saiga antelope. The saiga population distributed in Xinjiang, my country is almost extinct due to overhunting. In recent years, due to excessive poaching, hunting and illegal trade by humans, the population distributed in other Central Asian countries has also dropped sharply in just a few years. my country has listed the saiga tatarica in the first level of the "National Key Protected Wildlife List", and the "CITES" has listed the saiga tatarica in Appendix II.

[0003] The horns of the Gazelle (hereinafter referred to as antelope horns) are widely used in traditional Chinese medicine because of their efficacy in clearing away heat and detoxifying. In my country, the horns of the yellow sheep (Procapra gutturosa), Tibetan antelope (Procapra picticaudata), goitered gazelle (Gazella subgutturosa) and Tibetan antelope (Pantholops hodgsonii) can be used as substitutes for the horns of the Gazelle. In the Chinese medicinal materials market, antelope horns are made into powders, silk strips and pieces for sale. In addition, some vendors deliberately forge them, making it difficult to identify the types of antelope horns, which brings difficulties to the supervision of the legal medicinal use of antelope horns, and is not conducive to monitoring the poaching and smuggling of endangered species such as the Gazelle.

[0004] The existing methods for identifying antelope horns include morphological comparison, microstructure comparison, protein electrophoresis, thin-layer chromatography, etc. Compared with the above methods, DNA molecular technology is not limited by sample morphology and quantity, and the results are more accurate, which has unique advantages for its source identification. However, the current identification of antelope horn derived components mostly uses DNA barcoding combined with conventional PCR technology. Although this technology can qualitatively identify the species of antelope horns, this method has poor sensitivity, is time-consuming and labor-intensive, and has a long cycle.

[0005] Fluorescence quantitative PCR combines the characteristics of conventional PCR technology and spectral technology. It adds fluorescent groups to the ordinary PCR reaction system and uses the accumulation of fluorescent signals to monitor the entire PCR reaction process in real time. The entire reaction and detection are carried out in the reaction tube. After the PCR is completed, the system's own software calculates the Ct value of each sample and directly obtains the test result. This method omits the steps of electrophoresis, sequence determination and analysis after amplification. Together with nucleic acid extraction, the entire detection process can be completed within 2 hours. At the same time, this method has strong specificity, high sensitivity, and short time consumption. It also has a series of advantages such as no pollution, no toxicity, high throughput, multiple amplification, and quantitative analysis.

[0006] Therefore, the present invention designs to use fluorescent quantitative PCR to identify the horns of the saiga antelope and its products. Summary of the invention

[0007] One of the purposes of the present invention is to provide a primer-probe combination for identifying saiga antelope horns and products thereof, comprising a forward primer, a reverse primer and a probe, the sequences of which are as follows:

[0008] Forward primer F1_GB_COIII: 5'-TTACGGTTACCTTCTATGAGGC-3'

[0009] Reverse primer R1_GB_COIII: 5'-CTTGCTCCTACACCCGAAC-3'

[0010] Probe Prob_GBLY: FAM-5'-TAGATACTCCTGAGGCTAGAAGGACGGA-3'-BHQ1.

[0011] The second object of the present invention is to provide a kit for identifying saiga antelope horns and products thereof, comprising the above-mentioned primer-probe combination.

[0012] Furthermore, the kit also includes a positive quality control product, a negative quality control product and a fluorescence detection reagent.

[0013] The third object of the present invention is to provide the use of the above primer-probe combination or the above kit in identifying saiga antelope horns and their products.

[0014] A fourth object of the present invention is to provide a method for identifying whether a sample to be tested contains saiga antelope horns using the primer-probe combination or the kit, comprising the following steps:

[0015] Step 1, extracting DNA from the sample to be tested as a template;

[0016] Step 2, using the above primer-probe combination or the above kit to perform PCR amplification on the template of step 1, and collect the fluorescence signal;

[0017] Step 3: Determine whether there are saiga horns in the sample based on the fluorescence signal and Ct value calculated by the machine. A result with a Ct value greater than 35 is considered negative.

[0018] Furthermore, the reaction system for PCR amplification in step 2 is:

[0019]

[0020] The reaction conditions were as follows: react at 50°C for 2 min; react at 95°C for 30 s; react at 95°C for 10 s, and then react at 60°C for 40 s, repeated 40 times.

[0021] At present, the existing technology usually uses the mitochondrial cytochrome C oxidase subunit I (COⅠ) gene to identify antelope horns, while the present invention uses the mitochondrial cytochrome C oxidase III gene (COIII, ID: JN632700) as the target sequence, designs specific primer pairs and probes, and combines them with the fluorescent probe method to effectively distinguish between the horns of the Gazelle and its related species. It can effectively detect low-content products (such as medicinal powders and oral liquids containing the horns of the Gazelle) with high sensitivity.

[0022] Beneficial effects:

[0023] The standard curve regression equation of the fluorescence PCR method established in the present invention is: Y = -3.3266X + 39.655, and the correlation coefficient R 2 =0.9974, amplification efficiency Eff (%) = 99.824, the logarithm of the nucleic acid copy number and the Ct value of the reaction showed a good linear relationship, and the minimum nucleic acid copy number that could be amplified was 10 1 The sensitivity of the primer and probe combination of the present invention is 100 times higher than that of conventional PCR. The primer and probe combination of the present invention has strong specificity and can specifically amplify the components of the saiga antelope and accurately distinguish the saiga antelope horns and their products.

[0024] The TaqMan probe real-time fluorescence quantitative PCR method established by the present invention can achieve efficient amplification of the target fragment in a short time, and the entire reaction is simple and rapid, with high sensitivity, good repeatability, strong stability, and a wide range of application and promotion. The present invention can be applied to the rapid identification of relevant samples in cases involving saiga antelope horns and their products, and is particularly suitable for use in cases with large sample sizes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the fluorescence quantitative PCR amplification curve of the recombinant plasmid dilution.

[0026] Figure 2 It is the standard curve of fluorescence quantification of recombinant plasmid dilution.

[0027] Figure 3 It is a specific test of the fluorescence quantitative PCR system.

[0028] Figure 4 These are three photos of what appear to be saiga horns.

[0029] Figure 5 The fluorescence quantitative PCR amplification curves of three suspected saiga horns.

[0030] Figure 6 These are two photos of saiga horn products on sale in the market.

[0031] Figure 7These are the fluorescence quantitative PCR amplification curves of two saiga antelope horn products sold in the market. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] 1. Sensitivity test of fluorescence quantitative PCR reaction system

[0037] The sequence of the mitochondrial cytochrome C oxidase III gene (COIII, ID: JN632700) of the Gazelle was downloaded from GenBank, and a specific primer pair and probe were designed at 315-469 bp. The sequence is as follows:

[0038] Forward primer F1_GB_COIII: 5'-TTACGGTTACCTTCTATGAGGC-3'

[0039] Reverse primer R1_GB_COIII: 5'-CTTGCTCCTACACCCGAAC-3'

[0040] Probe Prob_GBLY: FAM-5'-TAGATACTCCTGAGGCTAGAAGGACGGA-3'-BHQ1.

[0041] The target fragment was 155 bp in length. After purification, the target fragment was connected to the pUC57 vector. The concentration of the recombinant plasmid was measured using a nucleic acid concentration meter. The copy number of the recombinant plasmid was calculated to be 3.92×10 10 The recombinant plasmid was diluted 10-fold to 10 1 . Each gradient of plasmid dilution (10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1The fluorescence quantitative PCR test was performed with 10 μL Probe qPCR SuperMix, 0.2 μL forward and reverse primers (10 μM), 0.4 μL probe (10 μM), 0.4 μL ROX, 2 μL DNA template, and 6.8 μL ddH2O. The reaction conditions were 95°C for 20 seconds, 95°C for 1 second, and then 60°C for 20 seconds, repeated 40 times. The amplification curve is shown in Figure 1 With Ct value as the ordinate and the template dilution multiple Log value as the abscissa, a standard curve is obtained, such as Figure 2 The standard curve regression equation is: Y = -3.3266X + 39.655, the correlation coefficient is R 2 =0.9974, amplification efficiency Eff(%) =99.824. The minimum detection amount of this method is 39.2 copies / μL, and it has a good linear relationship in a wide range.

[0042] 2. Specificity detection of fluorescent quantitative PCR reaction system

[0043] The established real-time fluorescence quantitative PCR method was used to amplify the nucleic acids of the horns of the Gazelle, its closely related species (the horns of the Tibetan antelope, the horns of the goat, and the horns of the yellow sheep), and other animal-derived products (cow horns and sheep horns) used to imitate the horns of the Gazelle, and a negative control was established.

[0044] The results are as follows Figure 3 As shown, except for the horns of the saiga antelope, the other samples did not show typical amplification curves, which indicated that the method had good specificity.

[0045] 3. Repeatability test of fluorescence quantitative PCR reaction system

[0046] The established real-time fluorescence quantitative PCR method was used to perform fluorescence quantitative PCR detection within and between groups using three positive DNA samples as templates. Three repeatability tests were performed. The results are shown in Table 1.

[0047] Table 1 Repeatability of fluorescence quantitative PCR reaction system

[0048]

[0049] In the repeatability test, the intra-group coefficient of variation was less than 1%, and the inter-group coefficient of variation was less than 2%, indicating that this method has good repeatability.

[0050] Example 2

[0051] Testing of suspected saiga horns and their products in related cases.

[0052] DNA extraction kit (Takara) was used to extract DNA from three suspected saiga antelope horns and their products ( Figure 4 ) to extract nucleic acid, and detect it using the real-time fluorescence quantitative PCR detection system established by the present invention. Figure 5 As shown, the samples to be tested all have typical amplification curves, and the Ct values ​​are less than 35.0, while the negative control has no typical amplification curve and no Ct value.

[0053] Example 3

[0054] Testing of 2 saiga horn products currently on the market.

[0055] DNA extraction kit (Takara) was used to extract DNA from two pieces of saiga antelope horn products on the market in the case. Figure 6 ) to extract nucleic acid, and detect it using the real-time fluorescence quantitative PCR detection system established by the present invention. Figure 7 As shown, antelope horn powder has a typical amplification curve with a Ct value less than 35.0; the Ct value of antelope horn oral liquid is greater than 35.0, and the content is relatively low; the negative control has no typical amplification curve and no Ct value.

Claims

1. A primer-probe combination for identifying saiga antelope horns and products thereof, characterized in that: It includes forward primer, reverse primer and probe, and its sequence is as follows: Forward primer F1_GB_COIII: 5'-TTACGGTTACCTTCTATGAGGC-3' Reverse primer R1_GB_COIII: 5'-CTTGCTCCTACACCCGAAC-3' Probe Prob_GBLY: FAM-5'-TAGATACTCCTGAGGCTAGAAGGACGGA-3'-BHQ1.

2. A kit for identifying saiga antelope horns and products thereof, characterized in that: The invention comprises the primer-probe combination according to claim 1.

3. The kit according to claim 2, characterized in that The kit also includes a positive quality control product, a negative quality control product and a fluorescence detection reagent.

4. Use of the primer-probe combination according to claim 1 or the kit according to claim 2 in identifying saiga antelope horns and products thereof.

5. A method for identifying whether a sample to be tested contains saiga antelope horns using the primer-probe combination of claim 1 or the kit of claim 2, characterized in that: The steps include: Step 1, extracting DNA from the sample to be tested as a template; Step 2, using the primer-probe combination of claim 1 or the kit of claim 2 to perform PCR amplification on the template of step 1, and collecting the fluorescent signal; Step 3: Determine whether there are saiga horns in the sample based on the fluorescence signal and Ct value calculated by the machine. A result with a Ct value greater than 35 is considered negative.

6. The method according to claim 5, characterized in that The reaction system for PCR amplification in step 2 is: The reaction conditions were as follows: react at 50°C for 2 min; react at 95°C for 30 s; react at 95°C for 10 s, and then react at 60°C for 40 s, repeated 40 times.

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