Primers, kits, and methods for rapid quantitative detection of characteristic nucleic acid molecules of Antrodia elata.

Abstract

The application discloses a characteristic nucleic acid molecule primer, a kit and a method for rapidly and quantitatively detecting Callophoma plicatus. The nucleic acid molecule primer is BSZ2F: 5'-CAAGTGCTCAACCGCTAC-3' and BSZ2R: 5'-TGCTGATTCTCCACGATG-3'. The application adopts a fluorescent quantitative PCR technology to detect, and the detection limit is as low as 0.03 ng / µL. The method is simple, specific, short in time and capable of being completed within 2 hours.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of rapidly detecting the authenticity of edible and medicinal fungi by fluorescent quantitative molecular technology, and particularly relates to a characteristic nucleic acid molecule primer, a kit and a method for rapidly and quantitatively detecting Ganoderma subresinosum. BACKGROUND

[0002] In New Edition of Chinese Materia Medica and Chinese Fungus Medicine and other works, Ganoderma subresinosum (Murrill) C.J. Humphrey is a medicinal fungus of Ganodermataceae in Polyporales, widely distributed in Guangdong, Hainan, Guangxi and Yunnan, and used as a medicine in some areas. The "Ganoderma subresinosum" used in China for many years is a polypore with a nearly black cap and white flesh, and the specific morphology is as follows: basidiocarp is annual, without stipe, and woody. The cap is semicircular or nearly fan-shaped, 6-15 cm long and 5-12 cm wide, black, with a lacquer-like luster and irregular wrinkles. The flesh is white, 0.3-1 cm thick. The tube is pale yellow, 0.9-1.4 cm long. The pore surface is pale white or slightly pale gray, and turns light brown after injury. The tube opening is nearly circular to angular, and the pore density is 4-6 per mm. The spores have two different sizes, the large spores are ellipsoidal, with a double-layer wall, colorless and transparent outer wall, smooth, colorless to light yellow-brown inner wall, 13-18 μm long and 9.5-12.5 μm wide, and the small spores are brown, spherical, with obvious small spines, 9-11 μm in diameter. In the species identification and classification research of the products named "Ganoderma subresinosum" sold in the market and used in some hospitals for a long time, it is found that there is confusion in the use of names, and the actual species sold is different, which leads to the fact that consumers are easy to buy wrong products, and the efficacy of the products cannot be guaranteed. The previous research also found that Ganoderma subresinosum is not the species of Ganoderma reported in some literatures, and its Latin name should be Ganoderma subresinosum (Murrill) C.J. Humphrey. However, the morphological identification is based on experience, and the appearance characteristics often have differences and changes; the traditional molecular amplification and sequencing identification are time-consuming and laborious. Therefore, it is of great significance to develop a technology for rapidly and quantitatively detecting the authenticity and content of Ganoderma subresinosum single or compound medicine, which is also of substantial application value for the commercial production of Ganoderma subresinosum products and the rapid inspection of traditional Chinese medicines. SUMMARY

[0003] The first object of the present application is to provide characteristic nucleic acid molecule primers capable of being used for rapidly detecting the authenticity of Ganoderma subresinosum and its related products and detecting the content of Ganoderma subresinosum.

[0004] The nucleic acid molecule primers include: 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 concentration of the genomic DNA of the sample to be detected, the nucleic acid molecule primers have extremely high binding specificity with Ganoderma subresinosum, and have lower or no specificity with Sanguinoderma rugosum (Blume & T. Nees) Y.F. Sun, D.H.Costa & B.K. Cui, G. gibbosum (Blume & T. Nees) Pat., G. sichuanense J.D. Zhao & X.Q. Zhang, G. tropicum (Jungh.) Bres., G. weberianum (Bres. & Henn. ex Sacc.) Steyaert, G. leucocontextum T.H. Li, W.Q. Deng, Sheng H. Wu, Dong M. Wang & H.P. Hu and G. lingzhi Sheng H. Wu, Y. Cao & Y.C. Dai. In the process of fluorescence quantitative PCR, the content of Ganoderma subresinosum DNA in the substrate is detected according to the strength of the fluorescence signal, and the sensitivity is high. Therefore, the authenticity of the fruiting bodies of Ganoderma subresinosum and its related products can be rapidly detected and the content can be determined by using the nucleic acid molecule primers through fluorescence quantitative PCR amplification.

[0005] The second object of the present application is to provide a kit for rapidly and quantitatively detecting Ganoderma subresinosum, which comprises the nucleic acid molecule primers BSZ2F and BSZ2R described above, and conventional DNA extraction reagents and fluorescence quantitative PCR reaction reagents.

[0006] The third object of the present application is to provide a method for rapidly and quantitatively detecting Ganoderma subresinosum, which uses the nucleic acid molecule primers BSZ2F and BSZ2R described above as amplification primers, takes the genomic DNA to be detected as a template, and rapidly detects Ganoderma subresinosum by using a fluorescence quantitative PCR method.

[0007] Preferably, the PCR amplification reaction system is 20 μL total volume: 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, template DNA 0.5 μL.

[0008] Preferably, the PCR amplification reaction procedure is 95℃ 30 s pre-denaturation; 95℃ 10 s, 62℃ 25 s, 40 cycles; 95℃ 15 s, 60℃ 60 s, 95℃ 15 s.

[0009] Based on the RBP2 gene sequence (SEQ ID NO. 1) of the Ganoderma boninense, the specific nucleic acid molecule primers BSZ2F and BSZ2R are designed, and the genomic DNA of the Ganoderma boninense is used as a template to perform PCR according to the conventional fluorescent quantitative PCR method (see Example 2 for details). The Ganoderma boninense can preferentially stimulate a fluorescent signal, and a specific gene fragment is obtained, while other Ganoderma or pseudogano derivatives such as Sichuan Ganoderma, tropical Ganoderma, and white Ganoderma have no Ct value (such as Figure 1 ) when the cycle number is less than 30, which shows that the nucleic acid molecule primers BSZ2F and BSZ2R of the present application have high amplification specificity and can obtain a specific gene fragment. The present application can be used for rapid detection of the authenticity of the fruiting bodies and related products of the Ganoderma boninense. The quantitative detection of the Ganoderma boninense is based on the standard curve of the specific RBP2 gene of the Ganoderma boninense (such as Figure 2 ), and the correlation coefficient is 0.9924, the linear range of the Ct value is 21.53-31.35, and the regression equation is y = -3.312x + 29.569. According to the regression equation, the relative content of the Ganoderma boninense sample can be calculated, and the quantitative detection of the fruiting bodies and related products of the Ganoderma boninense can be realized. The present application uses the fluorescent quantitative PCR technology for detection, and the detection limit is as low as 0.03 ng / μL. The method is simple, specific, and time-saving, and can be completed within 2 h. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The nucleic acid molecule BSZ2F and BSZ2R are used as primers to perform real-time fluorescent quantitative PCR amplification curve of different Ganoderma or pseudogano samples, as shown in Figure 1 , the A point is the Ct value of the Ganoderma boninense 22.96±0.35, and the fluorescent value reaches the threshold value, while other Ganoderma or pseudogano does not reach the threshold value in the whole fluorescent PCR cycle process (within 30 cycles).

[0011] Figure 2The standard curve of the specific gene of T. sublamellata was obtained by using BSZ2F and BSZ2R as primers and the DNA of the sample of T. sublamellata with different concentrations as a template according to the method of fluorescent quantitative PCR, the correlation coefficient was 0.9924, the linear range of Ct value was 21.53-31.35, and the regression equation was y = -3.312x + 29.569. According to the regression equation, the relative content of the sample of T. sublamellata to be measured can be calculated. DETAILED DESCRIPTION

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

[0013] Example 1: Extraction of genomic DNA of T. sublamellata

[0014] 0.1 g of the sample of T. sublamellata or the mixed sample containing T. sublamellata was weighed into a 1 mL EP tube, 2 sterile steel balls were put into the tube, and the sample was oscillated and ground in a sample grinder (LUKYM24, Guangzhou Luca) at 60 Hz for 180 s. The genomic DNA of T. sublamellata was extracted by using the HiPure HP Plant DNA Maxi Kit (D316303, Guangzhou Meiji Biological Technology Co., Ltd.) of Magen Company. The DNA concentration of the sample was determined by using a micro ultraviolet-visible spectrophotometer (NanoDrop One, Thermo Fisher Scientific), and all samples were uniformly diluted or concentrated to 50 ng / μL. The primers BSZ2F: 5'-CAAGTGCTCAACCGCTAC-3' and BSZ2R: 5'-TGCTGATTCTCCACGATG-3' and the fluorescent PCR reagent SYBR qPCR Master Mix (Q712-02, Nanjing Novozyme Biological Technology Co., Ltd.) were used, and the Real-time PCR reaction (QuantStudio 5, Thermo Fisher Scientific) was carried out by using the genomic DNA of T. sublamellata, P. pseudogigantea, G. subflavum, G. szechuanense, G. tropicum, G. weberianum, G. album and G. sinense with a concentration of 50 ng / μL as a template. The reaction system was 20 μL in total volume: 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, template DNA 0.5 μL. The reaction program was 95℃ 30 s pre-denaturation; 95℃ 10 s, 62℃ 25 s, 40 cycles; 95℃ 15 s, 60℃ 60 s, 95℃ 15 s. The results are shown in FIG. 1. Figure 1The fluorescence value of the thin tree mushroom reached the threshold value at the Ct average value of 22.96 (i.e. 23 PCR cycles), while the fluorescence values of other Ganoderma or false mushrooms did not reach the threshold value during the whole PCR cycle. The method can realize specific detection of the thin tree mushroom, and the characteristic nucleotide sequence of the thin tree mushroom is shown as SEQ ID NO. 1, and has 488 bases.

[0015] Example 2: Quantitative detection of DNA content of thin tree mushroom

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

[0017] Table 1 Ct values of thin tree mushroom DNA samples of different concentrations

[0018] Item No. 1 2 3 4 DNA concentration (logarithm to the base 10, lg) 2.50 1.50 0.71 -0.52 Ct value 21.53±0.01 24.15±0.04 27.37±0.30 31.35±0.30

[0019] The above is only a preferred embodiment of the present application, and it should be pointed out that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined by the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A primer for rapidly and quantitatively detecting a characteristic nucleic acid molecule of Poria vailantii, 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 Poria cocos, characterized in that, The nucleic acid molecule primer BSZ2F and BSZ2R in claim 1, and DNA extraction reagent and fluorescent quantitative PCR reaction reagent.

3. A method for rapid quantitative detection of Poria cocos, characterized in that, The nucleic acid molecule primer BSZ2F and BSZ2R in claim 1 are used as amplification primers, and the fluorescent quantitative PCR method is used to detect the Poria xylina.

4. The method of claim 3, wherein, The amplification reaction system of the fluorescent quantitative PCR method is 20 μL in total volume: 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 of claim 3, wherein, The amplification reaction procedure of the fluorescent quantitative PCR method is 95 ℃ for 30 s of pre-denaturation, 95 ℃ for 10 s, 62 ℃ for 25 s, 40 cycles, 95 ℃ for 15 s, 60 ℃ for 60 s, and 95 ℃ for 15 s.

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