Application of endogenous reference gene in rapid identification of Bauhinia purpurea

Abstract

The invention discloses an application of an endogenous reference gene in rapid identification of Bauhinia bauhinia, the nucleotide sequence of the endogenous reference gene is shown as SEQ ID NO.1. The method can be used for accurately distinguishing the Bauhinia bauhinia from similar easily-confused organisms such as rhododendron simsii, and qualitative and quantitative PCR methods for detecting the endogenous reference gene are respectively established by taking the endogenous reference gene as a target sequence. The method can provide a rapid detection basis for avoiding mistaken eating and misuse, is high in sensitivity, strong in specificity, simple to operate, low in cost and suitable for on-site real-time detection, and has a wide application prospect in rapid adulteration identification of similar species, and a reaction result is easy to observe.

Description

Technical Field

[0001] The invention relates to the technical field of biological species identification and nucleic acid in vitro amplification detection, and in particular to a detection method for rapidly identifying Bauhinia components based on internal standard genes and a kit thereof. Background Art

[0002] Bauhinia variegata, also known as Bauhinia, is a plant of the genus Bauhinia in the family Leguminosae. It mainly grows in bushes in sunny places in the mountains. There are more than 600 species worldwide, of which about 40 species are widely distributed in southern provinces of my country. It has strong adaptability and grows rapidly. Bauhinia variegata is mainly used as a landscape ornamental tree in many cities. In early spring, the flowers bloom before the leaves, and the tree is full of flowers, which is fresh and elegant, and has a high landscape ecological value. At the same time, Bauhinia variegata is also a common folk medicinal plant in Guangxi and Yunnan, my country. This is recorded in the "Chinese Materia Medica" and has high medicinal value. Its main variant, Bauhinia leucophylla, is also known as old white flower or jade lotus flower. It is edible and mostly grows on hillsides, roadsides, and around villages at an altitude of 800-1400 meters. Its flowers are rich in protein and carotene, and have the effects of clearing away heat and detoxifying, relieving cough and asthma, reducing phlegm and inflammation, and lowering blood lipids and blood pressure. It is a rare delicacy in Yunnan, Guangxi and other places. However, the white azalea, which is called "twin", is toxic. The large white azalea contains tetracyclic diterpenoid toxins, and the toxicity is mainly concentrated in the petals. It will have a certain impact on the body and mind of the poisoned person, and may even be fatal. Every year, there are countless poisoning incidents due to accidental ingestion and misuse. Therefore, while paying attention to the protection and development and utilization of Bauhinia leucophylla resources, it is extremely important to establish a sound and rapid and efficient food detection and identification system.

[0003] With the frequent occurrence of food safety problems such as adulteration of biological ingredients in food and accidental poisoning, people are increasingly eager to establish a corresponding detection and identification system to safeguard their legitimate rights and interests. In the past, there were morphological, physical and chemical, and omics identification technologies, and in the latter, there were modern molecular biological identification technologies. The latter, with more marker sites, higher polymorphism, and more efficient evaluation systems, is widely used in research such as germplasm resource evaluation, genetic diversity analysis, and kinship identification. It complements and gradually replaces traditional detection methods, and also provides solid technical support for adulteration identification in the food field. These technologies may require the use of multiple pairs of random primers for amplification before verification; or they may require the use of restriction endonucleases to digest the genome, and the results can be determined through electrophoresis fingerprinting and other technologies; or they may require sequencing and comparison of the products after ITS sequence amplification to achieve the purpose of detection.

[0004] The internal standard gene is a conservative DNA sequence with a constant copy number, species specificity, and no allele changes. It is a special genetic marker that distinguishes a specific species from other species. It can be used for the identification of species and their products and the detection of transgenic components. It must have both inter-species specificity and intra-species non-specificity. The selection of genes that are specific to the species but have low homology in other species is inter-species specificity. The internal standard genes of different cultivars or different tissue structures of the same species have high homology and similarity, which is intra-species non-specificity. In PCR detection, the most basic step is to detect the internal standard gene of the species, and during the detection, it is necessary to analyze whether the system establishment is reliable and whether the sample genomic DNA is suitable for PCR reaction. At the same time, genes with more complete sequences and fewer homologous sequences should be selected as internal standard genes of a species to ensure their specificity. Therefore, when a new internal standard gene of a species is developed, the gene information of the corresponding species should be searched through the database first, and BLAST comparison analysis should be performed to preliminarily screen out genes with fewer homologous sequences and specificity, and then the specificity of the primers should be verified by PCR, and finally primers with high sensitivity and good specificity should be selected as detection primers. The use of internal standard genes can be used to identify the source of food ingredients quickly and accurately. Bauhinia has high nutritional value and is popular among people, but poisoning incidents caused by accidental ingestion and misuse are endless, so identification through internal standard genes is very accurate and effective. Summary of the invention

[0005] The present invention provides a detection method for rapidly identifying Bauhinia components based on an internal standard gene. The nucleotide sequence of the internal standard gene is shown in SEQ ID NO: 1. The method of the present invention uses the internal standard gene as a target gene for species identification.

[0006] The Bauhinia species include white Bauhinia species and pink Bauhinia species.

[0007] During the detection of the present invention, specific primers capable of amplifying the internal standard gene are used, the nucleotide sequences of the specific primers are shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the detection is performed using a qualitative PCR method;

[0008] When the quantitative PCR method is used for detection, a probe is also included, and the nucleotide sequence of the probe is shown in SEQ ID NO:4.

[0009] Another object of the present invention is to provide a detection reagent or a kit comprising the above-mentioned specific primers and probes, wherein the detection reagent or the kit further comprises other conventional supporting detection reagents.

[0010] Advantages and technical effects of the present invention:

[0011] The method of the invention can be used to accurately distinguish similar and easily confused organisms such as Bauhinia and Rhododendron, and a qualitative and quantitative PCR method for detecting the internal standard gene is established with the internal standard gene as the target sequence, which can provide a rapid detection basis for avoiding accidental ingestion or misuse. The method of the invention has high sensitivity, strong specificity, simple operation, low cost, and easy observation of reaction results. It is suitable for on-site real-time detection and has broad application prospects in the rapid adulteration identification of similar species. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The electrophoresis results of DNA extraction of 9 different flower samples are shown in the figure: M is DNA Marker DL 2000; 1: pink azalea (Rhododendron simsii) flower sample; 2: pink azalea (Rhododendron simsii) leaf sample; 3: pink flower Bauhinia (Bauhinia variegata var.candida) flower sample; 4: white flower Bauhinia (Bauhiniaacuminata Linn) flower sample; 5: broom (Caragana rosea) flower sample 1; 6: broom (Caraganarosea) flower sample 2; 7: green manure flower (Astragalus sinicus) flower sample; 8: mulberry flower (Morus alba L.) flower sample; 9: mouse worm grass (Gnaphalium affine D.Don) flower sample;

[0013] Figure 2 The electrophoresis results of PCR amplification using 18s universal primers for 9 different flower samples, in the figure: M is DNAMarker DL 2000; 1-2: pink azalea (Rhododendron simsii) flower samples; 3-4: pink azalea (Rhododendron simsii) leaf samples; 5-6: pink flower Bauhinia (Bauhinia variegata var.candida) samples; 7-8: white flower Bauhinia (Bauhinia acuminata Linn) samples; 9-10: broom (Caragana rosea) sample 1; 11-12: broom (Caragana rosea) sample 2; 13-14: green manure flower (Astragalus sinicus) sample; 15-16: mulberry flower (Morusalba L.) sample; 17-18: mouse wormwood (Gnaphalium affine D.Don) sample; 0: negative control;

[0014] Figure 3The PCR amplification electrophoresis pattern of the specific verification result of the PCR candidate primers of the standard gene in Bauhinia scabra, in the figure: M is DNA Marker DL 2000, 0: negative control; 1-4: YTJ-1F / R; 5-8: YTJ-2F / R; 9-12: YTJ-3F / R; 13-16: YTJ-4F / R; 1-2, 5-6, 9-10, 13-14: Bauhinia variegata var. candida samples; 3-4, 7-8, 11-12, 15-16: Bauhinia acuminata Linn samples;

[0015] Figure 4 The results of species-specific qualitative verification of standard genes in Bauhinia. In the figure: M is DNA Marker DL2000; 1-2: pink azalea (Rhododendron simsii) flower samples; 3-4: pink azalea (Rhododendron simsii) leaf samples; 5-6: pink flower Bauhinia (Bauhinia variegata var.candida) samples; 7-8: white flower Bauhinia (Bauhinia acuminata Linn) samples; 9-10: broom (Caragana rosea) sample 1; 11-12: broom (Caragana rosea) sample 2; 13-14: green manure flower (Astragalus sinicus) sample; 15-16: mulberry flower (Morusalba L.) sample; 17-18: Gnaphalium affine D.Don sample; 0: negative control;

[0016] Figure 5 The results of species-specific quantitative verification of standard genes in Bauhinia, in the figure: 1-2: Bauhinia variegata var. candida samples; 3-4: Bauhinia acuminata Linn samples; 5-6: Rhododendron simsii flower samples; 7-8: Rhododendron simsii leaf samples; 9-10: Caragana rosea sample 1; 11-12: Caragana rosea sample 2; 13-14: Astragalus sinicus sample; 15-16: Morus alba L. sample; 17-18: Gnaphalium affine D. Don sample; 19: negative control;

[0017] Figure 6 It is the qualitative test result of the sensitivity of the standard gene primer detection in Bauhinia, where M is DNAMarker DL2000; 0: negative control; 1-2: 200 ng / μL; 3-4: 20 ng / μL; 5-6: 2 ng / μL; 7-8: 0.2 ng / μL; 9-10: 0.02 ng / μL; 11-12: 0.002 ng / μL; 13-14: 0.0002 ng / μL;

[0018] Figure 7 This is the quantitative detection amplification curve of the detection sensitivity of the standard gene primers in Bauhinia ovata;

[0019] Figure 8 This is the standard curve of the detection sensitivity of the standard gene primers in Bauhinia . DETAILED DESCRIPTION

[0020] The present invention is further explained by examples below, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified;

[0021] In the following examples, fresh pink azalea (Rhododendron simsii), pink Bauhinia (Bauhinia variegata var. candida), white Bauhinia (Bauhinia acuminata Linn), broom (Caraganarosea), green manure flower (Astragalus sinicus), mulberry flower (Morus alba L.), and Gnaphalium affine D. Don were all purchased from large local flower trading markets and large farmers' markets in Yunnan.

[0022] Example 1: Screening and verification of standard genes in Bauhinia ovata

[0023] 1. First, search for Bauhinia acuminata gene sequence information in the Nucleotide database of NCBI, and preliminarily screen out genes with few homologous sequences and species specificity through BLASTN comparison analysis. Perform multiple sequence specificity analysis on the gene fragments and DNA sequences of several other similar species. Finally, integrate the sequences and finally determine the gene fragment with the nucleotide sequence shown in SEQ ID NO:1 as the internal standard gene for the identification of Bauhinia acuminata.

[0024] 2. Design qualitative PCR and quantitative PCR primers and probes for the internal standard gene obtained through the IDT website, and perform BLAST comparison analysis on the designed PCR primers and probes in NCBI GenBank to verify their specificity;

[0025] Table 1 Sequences of universal primers, candidate primers and probes for PCR of Bauhinia ovata

[0026]

[0027] 2. Specific qualitative identification of standard genes in Bauhinia

[0028] The genomic DNA of 9 common flowers or tissues (Azalea rosa centifolia, Azalea rosa centifolia leaves, Bauhinia rosa centifolia, Bauhinia scabra, Broom 1, Broom 2, Green Manure Flower, Mulberry Flower, and Gnaphalium serratum) were extracted by CTAB method. The results of 1% agarose gel electrophoresis of the extracted DNA are shown in Figure 1 .

[0029] The DNA of nine flower species was amplified by PCR using the plant universal primer 18s-F / R (Table 1). Figure 2 ), the PCR reaction system is shown in Table 2, and the PCR amplification reaction program is: 95℃ pre-denaturation for 5min, 95℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 30s, 30 cycles, and 72℃ termination extension for 10min; then the designed primer pairs YTJ-1F / R, YTJ-2F / R, YTJ-3F / R and YTJ-4F / R (Table 1) were used to perform qualitative PCR amplification on the DNA of Bauhinia odorata and Bauhinia odorata, and the amplified products were analyzed by electrophoresis using 2% agarose gel ( Figure 3 );

[0030] Table 2 Qualitative PCR reaction system

[0031]

[0032] The results showed that the PCR amplification results were better when the primer pair YTJ-3F / R was used for amplification, the target band size was in line with expectations, and the electrophoresis bands were complete and bright, while the brightness was lighter when the primer pairs YTJ-1F / R and YTJ-2F / R were used for amplification, and non-specific amplification bands appeared in the negative control in the amplification with YTJ-4F / R. Therefore, YTJ-3F / R was selected as the specific primer for Bauhinia scabra in the PCR amplification system.

[0033] The selected primer pair YTJ-3F / R was used to perform qualitative PCR amplification on the DNA of 9 kinds of flowers or tissues, and the amplified products were analyzed by electrophoresis using 2% agarose gel. The PCR reaction system was the same as above. The PCR amplification reaction procedure was as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, 30 cycles, and 72°C termination extension for 10 min. The results are shown in Figure 4 As shown, the results showed that only the white flower Bauhinia and pink flower Bauhinia samples could amplify a 147bp fragment using primer pair YTJ-3F / R, which was consistent with the fragment size of the designed primers, while the other 7 flower tissue samples had no specific amplification bands, indicating that this gene and the corresponding primers can be used to specifically and qualitatively detect the components of Bauhinia.

[0034] 3. Specific quantitative identification of standard genes in Bauhinia

[0035] The primer pair YTJ-3F / R was used to perform SYBR Green I real-time quantitative PCR amplification detection with the genomic DNA of nine flower species as templates. The PCR reaction system is shown in Table 3 The SYBR Green I real-time fluorescence quantitative PCR two-step amplification program was as follows: pre-denaturation (95°C, 15 min); 40 cycles: denaturation (95°C, 10 s), annealing / extension (56°C, 30 s);

[0036] Table 3 SYBR Green I real-time fluorescence quantitative PCR reaction system verified by specific primers

[0037]

[0038] The results in Table 4 show that the Ct values ​​of SYBR Green I amplification of Bauhinia odorata and Bauhinia odorata samples were significantly lower than those of the negative control (Table 4), indicating that the gene and the corresponding primers can be used to specifically and quantitatively detect Bauhinia odorata components;

[0039] Table 4 Species-specific SYBR Green I amplification Ct values ​​of standard genes in Bauhinia ovale

[0040]

[0041]

[0042] Example 2: Sensitivity verification experiment of the qualitative PCR method of the present invention

[0043] The DNA of Bauhinia officinalis was selected as the test sample, and the sample genomic DNA was diluted tenfold to 200 ng / μL, 20 ng / μL, 2 ng / μL, 0.2 ng / μL, 0.02 ng / μL, 0.002 ng / μL, and 0.0002 ng / μL, respectively, for ordinary PCR amplification detection. The PCR reaction system was the same as Table 2. Figure 6 The results showed that when the DNA template content of Bauhinia hornbeam was 400ng, 40ng, 4ng, 0.4ng and 0.04ng, clear specific bands appeared on the electrophoresis diagram, and the brightness gradually weakened. When the template content dropped to 0.004ng, no amplification product was detected, which indicates that the detection limit of ordinary qualitative PCR is 0.04ng.

[0044] Example 3: Sensitivity verification experiment of the quantitative PCR method of the present invention

[0045] The DNA of Bauhinia officinalis was selected as the test sample, and the sample genomic DNA was diluted tenfold to 200 ng / μL, 20 ng / μL, 2 ng / μL, 0.2 ng / μL, 0.02 ng / μL, 0.002 ng / μL, and 0.0002 ng / μL, respectively, for Taqman fluorescent probe real-time quantitative PCR amplification detection (as shown in Table 5). The Taqman fluorescent probe real-time quantitative PCR amplification program (Fast mode) was as follows: 95°C pre-denaturation for 20 s, 95°C denaturation for 1 s, 56°C annealing for 20 s, 45 cycles, Figure 7 It can be seen that when the template amount is as low as 0.4ng, specific amplification can still occur, and the Ct value is significantly lower than that of the negative control, which indicates that the minimum detection limit of Taqman fluorescent probe quantitative PCR is 0.4ng. Figure 8 , the linear formula of Ct value and template starting amount can be obtained: y = -3.346x + 31.758, correlation coefficient R 2 is 0.996;

[0046] Table 5 Taqman probe real-time fluorescence quantitative PCR reaction system verified by specific primers

[0047]

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of an internal standard gene in rapid identification of Bauhinia ovata, wherein the nucleotide sequence of the internal standard gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: Specific primers for amplifying the internal standard gene according to claim 1 are used, and the nucleotide sequences of the specific primers are shown in SEQ ID NO: 2 and SEQ ID NO:

3.

3. The use according to claim 2, characterized in that: When quantitative detection is performed, a probe is also included, and its nucleotide sequence is shown in SEQ ID NO:

4.

4. A detection kit, characterized in that: Contains the specific primers used in the application described in claim 2.

5. A detection kit, characterized in that: Contains specific primers and probes used in the application described in claim 3.

Images