Characteristic nucleic acid molecular primer, kit and method for rapidly and quantitatively detecting ganoderma capense

Abstract

The invention discloses a characteristic nucleic acid molecular primer, a kit and a method for rapidly and quantitatively detecting ganoderma capense. The nucleic acid molecular primer is BSZ2F: 5 '-CAAGTGCTCAACCGCTAC-3', and the nucleic acid molecular primer is BSZ2F: 5 '-CAAGTGCTCAACCGCTAC-3'; and BSZ2R: 5 '-TGCTGATTTCCACGATG-3', and BSZ2R: 5 '- The fluorescent quantitative PCR technology is adopted for detection, the detection limit is as low as 0.03 ng / mu L, the method is simple, the specificity is good, the consumed time is short, and detection can be completed within 2 h.

Description

Technical Field

[0001] The invention belongs to the technical field of rapid detection of the authenticity of edible and medicinal fungi by means of fluorescent quantitative molecular technology, and particularly relates to characteristic nucleic acid molecule primers, a kit and a method for rapid quantitative detection of Ganoderma lucidum. Background Art

[0002] According to works such as "New Compilation of Chinese Materia Medica" and "Chinese Fungal Medicinal Materials," Ganoderma lucidum is sweet, slightly salty, and neutral in nature, and enters the heart, liver, spleen, lung, and kidney meridians. It can nourish the lungs and kidneys, harmonize the stomach and spleen, calm the mind and stabilize the spirit, strengthen the body's vitality, and also has therapeutic effects on the immune system. It is primarily used to treat conditions such as progressive muscular dystrophy, myotonia, lupus erythematosus, alopecia areata, and scleroderma. Ganoderma lucidum is a medicinal fungus of the Ganodermataceae family, Polyporales. It is widely distributed in Guangdong, Hainan, Guangxi, and Yunnan, and is used medicinally in some areas. Ganoderma lucidum, a polyporous fungus used for many years in China, has a nearly black cap and white flesh. Its morphology is characterized by an annual, sessile, woody basidiocarp. The cap is semicircular or nearly fan-shaped, 6 to 15 cm long and 5 to 12 cm wide, black in color, with a lacquer-like sheen and irregular wrinkles. The flesh is white and 0.3 to 1 cm thick. The tube is pale yellow and 0.9 to 1.4 cm long. The surface of the fungus pore is pale white or slightly pale gray, turning light brown after being injured; the tube mouth is nearly circular to angular, and the fungus pore density is 4 to 6 / mm. The basidiospores have two types of spores of different sizes. The megaspores are oval, double-walled, with a colorless, transparent, and smooth outer wall and inconspicuous small spines on the inner wall. They are colorless to light yellow-brown, 13 to 18 μm long and 9.5 to 12.5 μm wide; the microspores are brown, spherical, with obvious small spines, and a diameter of 9 to 11 μm. During the species identification and classification research on products sold under the trade name of "Bo Shu Zhi" and "Bo Shu Zhi" products used for a long time in some hospitals, it was found that there is confusion in the use of names, the fake is confused with the real, and the species actually sold vary greatly, making it very easy for consumers to buy the wrong products and the product efficacy cannot be guaranteed. Previous studies have also revealed that Ganoderma subresinosum is not the Ganoderma species reported in some literature; its Latin name should be Ganoderma subresinosum (Murrill) CJ Humphrey. However, morphological identification relies on experience, and appearance characteristics often vary. Traditional molecular amplification and sequencing identification are time-consuming and laborious. Therefore, the development of a rapid, quantitative method for detecting the authenticity and content of Ganoderma subresinosum alone or in combination with Ganoderma is of great significance to the healthy development of the Ganoderma subresinosum industry, and has substantial application value in the commercial production of Ganoderma subresinosum products and the rapid testing of Traditional Chinese Medicine. Summary of the Invention

[0003] The first object of the present invention is to provide characteristic nucleic acid molecule primers that can be used to quickly detect the authenticity of Ganoderma lucidum and its related products and to detect the content of Ganoderma lucidum.

[0004] The nucleic acid molecule primer comprises: BSZ2F: 5'-CAAGTGCTCAACCGCTAC-3', BSZ2R: 5'-TGCTGATTCTCCACGATG-3'. The annealing temperatures of the nucleic acid molecule primer sequences BSZ2F and BSZ2R are similar. Under the same genomic DNA concentration of the test sample, the nucleic acid molecule primer has extremely high binding specificity to Ganoderma subresinosum, but has low or no specificity to Sanguinoderma rugosum (Blume & T.Nees) YFSun, DHCosta & B.K.Cui, G.gibbosum (Blume & T.Nees) Pat., G.sichuanense JDZhao & X.Q.Zhang, G.tropicum (Jungh.) Bres., G.weberianum (Bres. & Henn.exSacc.) Steyaert, G.leucocontextum THLi, WQDeng, Sheng H.Wu, DongM.Wang & H.P.Hu and G.lingzhi Sheng H.Wu, Y.Cao & Y.C.Dai. During the fluorescence quantitative PCR process, the intensity of the fluorescent signal is used to detect the DNA content of the G. thunbergii in the substrate with high sensitivity. Therefore, using the nucleic acid molecule primers through fluorescence quantitative PCR amplification can quickly detect the authenticity of G. thunbergii fruiting bodies and related products and determine their content.

[0005] The second object of the present invention is to provide a kit for rapid quantitative detection of Ganoderma lucidum, which comprises the above-mentioned nucleic acid molecule primers BSZ2F and BSZ2R, as well as conventional DNA extraction reagents and fluorescent quantitative PCR reaction reagents.

[0006] The third object of the present invention is to provide a method for rapid quantitative detection of Ganoderma lucidum, which uses the above-mentioned nucleic acid molecule primers BSZ2F and BSZ2R as amplification primers, takes the genome to be tested as a template, and uses fluorescent quantitative PCR method to rapidly detect Ganoderma lucidum.

[0007] Preferably, the PCR amplification reaction system has a total volume of 20 μL: SYBR qPCR Master Mix 10.0 μL, ddH2O 8.5 μL, BSZ2F 10 μmol / L 0.5 μL, BSZ2R 10 μmol / L 0.5 μL, and template DNA 0.5 μL.

[0008] Preferably, the PCR amplification reaction procedure is as follows: pre-denaturation at 95°C for 30s; 40 cycles of 95°C for 10s, 62°C for 25s; 95°C for 15s, 60°C for 60s, and 95°C for 15s.

[0009] Based on the RBP2 gene sequence of Ganoderma lucidum (SEQ ID NO. 1), the present invention designed specific nucleic acid molecule primers BSZ2F and BSZ2R, and used the genomic DNA of Ganoderma lucidum as a template, and performed PCR according to a conventional fluorescence quantitative PCR method (see Example 2 for details). Ganoderma lucidum can preferentially excite the fluorescent signal to obtain a specific gene fragment, while other Ganoderma lucidum or pseudo-Ganoderma lucidum samples such as Sichuan Ganoderma lucidum, tropical Ganoderma lucidum, and white Ganoderma lucidum have no Ct value when the cycle number is less than 30 (such as Figure 1 ), showing that the nucleic acid molecule primers BSZ2F and BSZ2R of the present invention have high amplification specificity and can obtain specific gene fragments. The present invention can be used to quickly detect the authenticity of the fruiting bodies of Ganoderma lucidum and its related products. The quantitative detection of Ganoderma lucidum is based on the standard curve of the Ganoderma lucidum specific RBP2 gene (such as Figure 2 ), with a correlation coefficient of 0.9924, a Ct value linear range of 21.53-31.35, and a regression equation of y = -3.312x + 29.569. Based on this regression equation, the relative content of the tested sample of Gastrodia elata can be calculated, enabling quantitative detection of Gastrodia elata fruiting bodies and related products. The present invention uses fluorescent quantitative PCR technology for detection, with a detection limit as low as 0.03 ng / μL. The method is simple, has good specificity, and is quick to complete, taking less than 2 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The amplification curves of real-time fluorescence quantitative PCR of different Ganoderma lucidum or pseudo-Ganoderma lucidum samples were obtained by using nucleic acid molecules BSZ2F and BSZ2R as primers according to the fluorescence quantitative PCR method. Figure 1 As shown, point A is the Ct value of Ganoderma lucidum 22.96±0.35, and the fluorescence value reaches the threshold, while other Ganoderma lucidum or Pseudo-Ganoderma lucidum did not reach the threshold during the entire fluorescence PCR cycle process (within 30 cycles).

[0011] Figure 2 Using DNA from Gastrodia elata samples of varying concentrations as templates and primers BSZ2F and BSZ2R using quantitative PCR, a standard curve for the Gastrodia elata-specific gene was generated. The curve had a correlation coefficient of 0.9924, a linear Ct range of 21.53-31.35, and a regression equation of y = -3.312x + 29.569. This regression equation can be used to calculate the relative content of the Gastrodia elata sample. DETAILED DESCRIPTION

[0012] According to the following examples, those skilled in the art can better understand the present invention. The examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0013] Example 1: Extraction of Ganoderma lucidum genomic DNA

[0014] Weigh 0.1 g of G. thunbergii sample or a mixed sample containing G. thunbergii into a 1 mL EP tube. Two sterile steel balls were placed in a sample grinder (Guangzhou Luka, model LUKYM24) and ground at 60 Hz for 180 s. Genomic DNA from G. thunbergii was extracted using the Magen HiPure HP Plant DNA Maxi Kit (Guangzhou Meiji Biotechnology Co., Ltd., number D316303). DNA concentration was determined using a micro-UV-visible spectrophotometer (NanoDrop One, Thermo Fisher Scientific). All samples were diluted or concentrated to 50 ng / μL. Real-time PCR reactions (QuantStudio 5, Thermo Fisher Scientific) were performed using primers BSZ2F: 5′-CAAGTGCTCAACCGCTAC-3′ and BSZ2R: 5′-TGCTGATTCTCCACGATG-3′ and the fluorescent PCR reagent SYBR qPCR Master Mix (Nanjing Novozymes Biotech Co., Ltd., No. Q712-02). Genomic DNA from Ganoderma lucidum, Ganoderma sutsugamushi, Ganoderma lucidum pedunculates, Ganoderma sichuanensis, Ganoderma tropicalis, Ganoderma webii, Ganoderma lucidum white flesh, and Ganoderma lucidum at a concentration of 50 ng / μL was used as template. The reaction system was a total volume of 20 μL: SYBR qPCR Master Mix 10.0 μL, ddH2O 8.5 μL, BSZ2F (10 μmol / L) 0.5 μL, BSZ2R (10 μmol / L) 0.5 μL, and template DNA 0.5 μL. The reaction program was pre-denaturation at 95°C for 30s; 40 cycles of 95°C for 10s, 62°C for 25s; 95°C for 15s, 60°C for 60s, and 95°C for 15s. Figure 1 The fluorescence value of Ganoderma lucidum reached the threshold at an average Ct value of 22.96 (i.e., after 23 PCR cycles); however, other Ganoderma lucidum and Pseudoglossi lucidum did not reach the threshold value throughout the entire PCR cycle. This method enables the specific detection of Ganoderma lucidum. The characteristic nucleotide sequence of Ganoderma lucidum is shown in SEQ ID NO. 1, which consists of 488 bases.

[0015] Example 2: Quantitative detection of DNA content of Ganoderma lucidum

[0016] Using known different concentrations of Ganoderma lucidum genomic DNA samples as templates, BSZ2F and BSZ2R were used as primers to amplify according to the fluorescent quantitative PCR method of Example 1. The Ct values ​​of the Ganoderma lucidum specific gene fragment SEQ ID NO.1 were obtained (see Table 1), and a standard curve was drawn (see Figure 2 ). Its correlation coefficient R 2 =0.9924, the linear range of Ct value (<35) is 21.53-31.35, and the regression equation is y=-3.312x+29.569. According to this regression equation, the relative content of the tested sample of Ganoderma lucidum can be calculated. According to the above-mentioned fluorescence quantitative PCR method, the minimum detection amount test was carried out, and the results showed that the detection limit was as low as 0.3ng / mL (Ct<35). Quantitative detection of Ganoderma lucidum can be achieved.

[0017] Table 1 Ct values ​​of Ganoderma lucidum DNA samples with different concentrations

[0018]

[0019] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

[0020] SEQ ID NO.1

[0021]

Claims

1. A characteristic nucleic acid molecule primer for rapid quantitative detection of Ganoderma lucidum, characterized in that: The nucleic acid molecule primer is: BSZ2F: 5′-CAAGTGCTCAACCGCTAC-3′; BSZ2R: 5′-TGCTGATTCTCCACGATG-3′.

2. A kit for rapid quantitative detection of Ganoderma lucidum, characterized in that: Comprising the nucleic acid molecule primer according to claim 1 BSZ2F and BSZ2R, as well as conventional DNA extraction reagents and fluorescence quantitative PCR reaction reagents.

3. A method for rapid quantitative detection of Ganoderma lucidum, characterized in that: Using the nucleic acid molecule primer according to claim 1 BSZ2F and BSZ2R were used as amplification primers, and the genome to be tested was used as a template to detect Ganoderma lucidum using the fluorescence quantitative PCR method.

4. The method according to claim 3, characterized in that The PCR amplification reaction system has a total volume of 20 μL: SYBR qPCR Master Mix 10.0 μL, ddH2O 8.5 μL, BSZ2F 10 μmol / L 0.5 μL, BSZ2R 10 μmol / L 0.5 μL, and template DNA 0.5 μL.

5. The method according to claim 3, characterized in that: The PCR amplification has a reaction procedure of pre-denaturation at 95°C for 30s; 95°C for 10s, 62°C for 25s, 40 cycles; 95°C for 15s, 60°C for 60s, and 95°C for 15s.

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