Primer group for detecting spores and hyphae of beauveria bassiana and application of primer group
By designing specific primer sets for the A5201 gene and A1176 gene of Coccidioidae, combined with RT-qPCR technology, efficient detection and expression level analysis of the spores and mycelium of Coccidioidae are achieved, solving the problem that is difficult to effectively detect in the existing technology, and providing an in-depth understanding of the biology and ecological functions of Coccidioidae.
Patent Information
- Application Number
- CN202411720669.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-03-11
AI Technical Summary
The existing technology is difficult to effectively detect the existence and expression levels of spores and mycelium of Cocciferus, which affects the understanding and utilization of biological characteristics, ecological functions and application value of Cocciferus.
A primer set for detecting spores and mycelium of C. leucorrhizalis was designed, including specific primers for the A5201 gene and A1176 gene, and quantitatively detected by RT-qPCR technology to achieve the analysis of specific amplification and expression levels of spores and mycelium.
This technical method can efficiently and specifically detect the expression levels of spores and mycelium of Coccidioidae, providing a non-disease diagnostic and treatment tool to help in-depth understanding of the biological characteristics and ecological functions of Coccidioidae.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer set for detecting spores and hyphae of Beauveria bassiana and an application thereof. Background Art
[0002] Beauveria bassiana is an entomogenous fungus with a wide host range. It belongs to the subphylum Deteromyeotina, class Hyphomycetes, order Hyphomycetales, family Moniliaceac, and genus Beauveria. According to relevant records, it can parasitize about 700 species of insects in 15 orders and 149 families, and more than 10 species of ticks and mites in 6 families of the order Acarina. The genus Beauveria was established in 1912, and nearly 19 species have been described. It was not until 1954 that Macleod confirmed that the genus had only two species, one is Beauveria bassiana, and the other is Beauveria bassiana.
[0003] Under suitable temperature and humidity conditions, the conidia of Beauveria bassiana first absorb water and swell, and can germinate as soon as possible after inoculation. As the number of germinated spores gradually increases, the germination peak is reached after 15 hours. Under certain conditions, the germinated conidia or mycelium can produce cylindrical, pear-shaped or elliptical budspores or cylindrical arthrospores, and some arthrospores are composed of several cells. Spherical or elliptical endospores can also be formed inside the mycelium, occurring singly or in clusters, and sometimes simultaneously with arthrospores. Therefore, Beauveria bassiana can reproduce asexually through a variety of methods such as conidia, budspores, arthrospores, endospores and the breakage of mycelium, and the type of life history belongs to the polyspore type. The colony of Beauveria bassiana is velvety, tufted to powdery, and the color is white or light yellow, occasionally pink. Aerial hyphae are transparent, smooth, and grow in flocculent form. Conidiophores are mostly grown on hyphae, often densely clustered, but sometimes in whorls or solitary. Conidial cells and vesicles often proliferate, gathering into spherical to ovoid dense spore heads on conidiophores and hyphae. Conidia are monospores, transparent, thin-walled, without thick-walled spores.
[0004] As the two basic components of Beauveria bassiana, spores and hyphae play a pivotal role. They are not only the key links in the life cycle of Beauveria bassiana, but also an important window for understanding the biological characteristics, ecological functions and application value of Beauveria bassiana. Summary of the invention
[0005] The present invention aims to provide a primer set for detecting spores and hyphae of Beauveria bassiana and application thereof.
[0006] To achieve the above object, the present invention adopts the following technical solution: A primer set for detecting spores and hyphae of Beauveria bassiana, the primer set comprising primers for detecting spores of Beauveria bassiana and primers for detecting hyphae of Beauveria bassiana; the primers for detecting spores of Beauveria bassiana are primers for detecting the A5201 gene of Beauveria bassiana, and the nucleotide sequence of the A5201 gene is shown in SEQ ID NO.7; the primers for detecting hyphae of Beauveria bassiana are primers for detecting the A1176 gene of Beauveria bassiana, and the nucleotide sequence of the A1176 gene is shown in SEQ ID NO.1; Furthermore, the nucleotide sequences of the primers for detecting the Beauveria bassiana A5201 gene are: upstream primer 5′-ATGAGCACTTTGAGCTTCGAC-3′, downstream primer 5′-TTATCTTCTCCGTGGGCTGTC-3′; the nucleotide sequences of the primers for detecting the Beauveria bassiana A1176 gene are: upstream primer 5′-ATGAAGACTGCTGTTGGTATTGTTT-3′, downstream primer 5′-TCATATTTCTCGCGAGAAACGAC-3′.
[0007] The above-mentioned primer set is used in detecting the transcription levels of the A5201 gene and the A1176 gene of Beauveria bassiana for non-disease diagnosis and treatment purposes. The nucleotide sequence of the A5201 gene is shown in SEQ ID NO.7, and the nucleotide sequence of the A1176 gene is shown in SEQ ID NO.1.
[0008] The application of the above primer set in quantitative detection of Beauveria bassiana spores and hyphae is for non-disease diagnosis and treatment purposes.
[0009] A method for detecting the transcription levels of A5201 gene and A1176 gene of Beauveria bassiana by RT-qPCR, the method being for non-disease diagnosis and treatment purposes, comprising the steps of adding the above-mentioned primers to a reaction system to perform single-plex real-time fluorescence quantitative PCR amplification; Furthermore, the reaction system of the single-plex real-time fluorescence quantitative PCR amplification is: 1 μl of cDNA template, 1 μl of upstream primer, 1 μl of downstream primer, Hieff TM qPCR SYBR Green Master Mix 25 μl, sterile ultrapure water 22 μl; Furthermore, the reaction conditions for the single-plex real-time fluorescence quantitative PCR amplification are: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing / extension at 60°C for 30 s, and 35 cycles; and the melting curve analysis conditions are: 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1:The electrophoresis results of the common PCR products of A1176 gene with fluorescent quantitative primers. Lanes 1 to 2 are pure spore samples, and lanes 3 to 4 are pure mycelium samples. The marker bands are 5000, 3000, 2000, 1000, 750, 500, 250, and 100 bp from top to bottom.
[0011] Figure 2 :The electrophoresis results of the common PCR products of A5201 gene with fluorescent quantitative primers. Lanes 1 to 2 are pure spore samples, and lanes 3 to 4 are pure mycelium samples. The marker bands are 5000, 3000, 2000, 1000, 750, 500, 250, and 100 bp from top to bottom.
[0012] Figure 3 : Expression of A1176 gene in pure mycelium of Beauveria bassiana at different culture times.
[0013] Figure 4 : Standard curve of pure mycelium dry weight of Beauveria bassiana-A1176 gene CT value.
[0014] Figure 5 : Expression of A5201 gene in pure spores of Beauveria bassiana at different culture times.
[0015] Figure 6 : Results of RT-qPCR and blood cell counting plate test of pure spores of Beauveria bassiana.
[0016] Figure 7 : Standard curve of CT value of Beauveria bassiana A5201 gene-spore count.
[0017] Figure 8 : Quantitative detection results of hyphae-spore mixture of Beauveria bassiana. DETAILED DESCRIPTION
[0018] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.
[0019] The Beauveria bassiana BbFZ-51 involved in the following embodiments is provided by the Fujian Academy of Forestry Sciences, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, and is classified and named Beauveria bassiana, with a deposit number of CGMCC NO.41505, a deposit date of September 9, 2024, and a deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and a postal code of 100101.
[0020] Embodiment 1: S1: Take 200g of peeled potatoes, add 1000ml of water, boil for 30min, filter with gauze, then weigh 20g of glucose and 20g of agar powder and add them to the filtrate, distilled water to 1L, sterilize at 121℃ for 30min to obtain PDA solid culture medium; spread the sterilized cellophane on the PDA solid culture medium, then inoculate the BbFZ-51 bacterial block of Beauveria bassiana on the cellophane under sterile conditions, culture at 28℃ for 5 days, scrape the mycelium on the cellophane, freeze it with liquid nitrogen and store it in a -80℃ refrigerator as a pure mycelium sample; spread the sterilized cellophane on the PDA solid culture medium, then inoculate the BbFZ-51 bacterial block of Beauveria bassiana on the cellophane under sterile conditions, culture at 28℃ for 20 days, form spores, collect the surface spore layer, freeze it with liquid nitrogen and store it in a -80℃ refrigerator as a pure spore sample.
[0021] S2: RNA was extracted from pure mycelium samples and pure spore samples of Beauveria bassiana BbFZ-51, and transcriptome sequencing was performed. The data obtained by sequencing were sorted and analyzed, and 7 differentially expressed genes were obtained, namely A1176, A6321, A2145, A2378, A3127, A4439, and A5201 (Table 1). Among them, the nucleotide sequence of the A1176 gene is shown in SEQ ID NO.1, the nucleotide sequence of the A6321 gene is shown in SEQ ID NO.2, the nucleotide sequence of the A2145 gene is shown in SEQ ID NO.3, the nucleotide sequence of the A2378 gene is shown in SEQ ID NO.4, the nucleotide sequence of the A3127 gene is shown in SEQ ID NO.5, the nucleotide sequence of the A4439 gene is shown in SEQ ID NO.6, and the nucleotide sequence of the A5201 gene is shown in SEQ ID NO.7, wherein the A1176 and A6321 genes are pure mycelium-specific expression genes of Beauveria bassiana BbFZ-51, and the A2145, A2378, A3127, A4439, and A5201 genes are pure spore-specific expression genes of Beauveria bassiana BbFZ-51.
[0022] Table 1 Expression information of differentially expressed genes in pure mycelium and pure spore samples of Beauveria bassiana BbFZ-51 (read counts) Pure Mycelium-1 Pure Mycelium-2 Pure Mycelium-3 Pure Spore-1 Pure Spore-2 Pure Spore-3 A1176 gene 1155 751 1163 0 0 0 A6321 gene 827 542 754 1 0 1 A2145 gene 3 2 0 853 905 851 A2378 gene 1 0 1 5637 5881 5915 A3127 gene 21 0 16 3162 3221 3212 A4439 gene 0 1 1 2425 2556 2572 A5201 gene 0 0 0 3283 3351 3251 S3: Based on the nucleotide sequences of the seven differentially expressed genes, fluorescent quantitative primers were designed using bioinformatics software, and Fuzhou Shangya Biotechnology Co., Ltd. was commissioned to synthesize the primers (Table 2). Genomic DNA was extracted from pure mycelial samples and pure spore samples of Beauveria bassiana BbFZ-51, and conventional PCR amplification was performed using fluorescent quantitative primers of genes A1176, A6321, A2145, A2378, A3127, A4439, and A5201. The conventional PCR amplification system included: 2 μl DNA template, 1 μl 10 μM upstream primer, 1 μl 10 μM downstream primer, 10 μl 2×Taq PCR MasterMix, and 6 μl ddH2O. The conventional PCR amplification conditions were: 93°C pre-denaturation for 3 min; 93°C denaturation for 45 s, 55°C annealing for 45 s, 72°C extension for 1.5 min, 30 cycles; 72°C extension for 10 min. The amplified products were detected by agarose gel electrophoresis. The results showed that primers A1176-F / A1176-R could specifically amplify pure mycelium samples of Beauveria bassiana BbFZ-51, with a product length of 606 bp, while no bands were amplified for pure spore samples of Beauveria bassiana BbFZ-51 ( Figure 1 ); Primers A5201-F / A5201-R can specifically amplify pure spore samples of Beauveria bassiana BbFZ-51, with a product length of 540 bp, but no band can be amplified for pure mycelium samples of Beauveria bassiana BbFZ-51 ( Figure 2 ); other primers could not amplify bands for pure mycelium samples and pure spore samples of Beauveria bassiana BbFZ-51. Therefore, primers A1176-F / A1176-R and A5201-F / A5201-R were subsequently selected to establish the RT-qPCR detection method.
[0023] Table 2 Fluorescence quantitative primers for differentially expressed genes S3: The Bb-actin gene was selected as the internal reference. The internal reference primers have been published in [CHO EM, KIRKLAND BH, HOLDERD J, et al. Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana [J]. Microbiology, 2007, 153 (10): 3438–3447.], and the sequences are: F: 5'-TTGGTGCGAAACTTCAGCGTCTAGTC-3', R: 5'-TCCAGCAAATGTGGATCTCCAAGCAG-3'. The total RNA of pure mycelial samples and pure spore samples of Beauveria bassiana BbFZ-51 was extracted using an RNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd.), and the corresponding cDNA was obtained as a template using oligo (dT) primers for reverse transcription. RT-qPCR was performed using primers A1176-F / A1176-R and A5201-F / A5201-R, respectively. The RT-qPCR reaction system contained: 1 μl of cDNA template, 1 μl of 10 μM upstream primer, 1 μl of 10 μM downstream primer, and Hieff TM qPCR SYBR Green Master Mix (Low Rox Plus) (Yisheng Biotechnology (Shanghai) Co., Ltd.) 25μl, sterile ultrapure water 22μl. RT-qPCR reaction conditions are: 94℃ pre-denaturation for 2min; 94℃ denaturation for 15s, 60℃ annealing / extension for 30s, 35 cycles; melting curve analysis conditions are: 95℃ 15s, 60℃ 1min, 95℃ 15s. After the RT-qPCR reaction, 2 -ΔCt The relative expression levels of A1176 and A5201 genes were calculated by the method. The results are shown in Table 3.
[0024] Table 3 RT-qPCR test results Gene samples Pure Mycelium-1 Pure Mycelium-2 Pure Mycelium-3 Pure Spore-1 Pure Spore-2 Pure Spore-3 A1176 26.608 26.115 28.477 / / / A5201 / / / 24.112 24.186 11.623 Note: “ / ” means Undetermined.
[0025] Embodiment 2: S1: Take 200g of peeled potatoes, add 1000ml of water, boil for 30min, filter with gauze, then weigh 20g of glucose and 20g of agar powder and add them to the filtrate, dilute to 1L with distilled water, sterilize at 121℃ for 30min to obtain PDA solid culture medium; spread the sterilized cellophane on the PDA solid culture medium, and then inoculate the BbFZ-51 bacterial block of Beauveria bassiana on the cellophane under sterile conditions, culture at 28℃ for 12h, 24h, 36h, and 48h, respectively, and scrape the mycelium on the cellophane, quickly freeze with liquid nitrogen, and store in a -80℃ refrigerator as a pure mycelium sample. Use RNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd.) to extract total RNA, use oligo(dT) primer for reverse transcription to obtain the corresponding cDNA as a template, and use primers A1176-F / A1176-R for RT-qPCR. The Bb-actin gene was selected as the internal reference. The internal reference primers were published in [CHO EM, KIRKLAND BH, HOLDERD J, et al. Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana [J]. Microbiology, 2007, 153 (10): 3438–3447.], and the sequences were: F: 5'-TTGGTGCGAAACTTCAGCGTCTAGTC-3', R: 5'-TCCAGCAAATGTGGATCTCCAAGCAG-3'. The RT-qPCR reaction system included: 1 μl of cDNA template, 1 μl of 10 μM upstream primer, 1 μl of 10 μM downstream primer, and Hieff TM qPCR SYBRGreen Master Mix (Low Rox Plus) (Yisheng Biotechnology (Shanghai) Co., Ltd.) 25μl, sterile ultrapure water 22μl. RT-qPCR reaction conditions are: 94℃ pre-denaturation for 2min; 94℃ denaturation for 15s, 60℃ annealing / extension for 30s, 35 cycles; melting curve analysis conditions are: 95℃ 15s, 60℃ 1min, 95℃ 15s. After the RT-qPCR reaction is completed, 2 -ΔCt The relative expression of A1176 gene was calculated by the method. The results showed that ( Figure 3 ), with the extension of culture time, the expression level of A1176 gene in pure mycelium of Beauveria bassiana BbFZ-51 first increased and then decreased, which was basically consistent with the change trend of the expression level of the transcriptome. This provides important basic data for in-depth research on the A1176 gene of Beauveria bassiana.
[0026] At the same time, the obtained pure mycelium samples of Beauveria bassiana BbFZ-51 were placed in a 45°C drying oven and dried to constant weight to obtain the pure mycelium dry weight. A standard curve was drawn with the pure mycelium dry weight (mg) of Beauveria bassiana BbFZ-51 as the abscissa and the CT value as the ordinate, and the standard curves of the pure mycelium samples taken after 12h, 24h, 36h, and 48h of culture were obtained as shown in the figure. Figure 4 The linear regression equation corresponding to the standard curve of the pure mycelium sample taken after 12 hours of culture is: y = -12.977x + 425.35 (R 2 =0.999), the linear regression equation corresponding to the standard curve of the pure mycelium sample taken after 24h of culture is: y = -14.959x + 465.54 (R 2 =0.9967), and the linear regression equation corresponding to the standard curve of the pure mycelium sample taken after 36 hours of culture was: y = -18.818x + 552.71 (R 2 =0.9601), the linear regression equation corresponding to the standard curve of the pure mycelium sample taken after 48h of culture is: y = -9.9921x + 300.11 (R 2 =0.9838).
[0027] S2: Take 200g of peeled potatoes, add 1000ml of water, boil for 30min, filter with gauze, then weigh 20g of glucose and 20g of agar powder and add them to the filtrate, distilled water to 1L, sterilize at 121℃ for 30min to obtain PDA solid culture medium; spread the sterilized cellophane on the PDA solid culture medium, and then inoculate the BbFZ-51 bacterial block of Beauveria bassiana on the cellophane under sterile conditions, culture at 28℃ for 48h, 60h, 96h, 168h, and 216h, respectively, collect the surface spore layer, freeze it with liquid nitrogen and store it in a -80℃ refrigerator as a pure spore sample, use RNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd.) to extract total RNA, use oligo(dT) primer for reverse transcription to obtain the corresponding cDNA as a template, and use primers A5201-F / A5201-R for RT-qPCR. The Bb-actin gene was selected as the internal reference. The internal reference primers were published in [CHO EM, KIRKLAND BH, HOLDER DJ, et al. Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana [J]. Microbiology, 2007, 153 (10): 3438–3447.], and the sequences were: F: 5'-TTGGTGCGAAACTTCAGCGTCTAGTC-3', R: 5'-TCCAGCAAATGTGGATCTCCAAGCAG-3'. The RT-qPCR reaction system included: 1 μl of cDNA template, 1 μl of 10 μM upstream primer, 1 μl of 10 μM downstream primer, and Hieff TM qPCR SYBRGreen Master Mix (Low Rox Plus) (Yisheng Biotechnology (Shanghai) Co., Ltd.) 25μl, sterile ultrapure water 22μl. RT-qPCR reaction conditions are: 94℃ pre-denaturation for 2min; 94℃ denaturation for 15s, 60℃ annealing / extension for 30s, 35 cycles; melting curve analysis conditions are: 95℃ 15s, 60℃ 1min, 95℃ 15s. After the RT-qPCR reaction is completed, 2 -ΔCt The relative expression of A5201 gene was calculated by the method. The results showed that ( Figure 5 ), with the extension of culture time, the expression level of A5201 gene in pure spores of Beauveria bassiana BbFZ-51 gradually increased, which was basically consistent with the change trend of expression level in the transcriptome. This provides important basic data for in-depth research on the A5201 gene of Beauveria bassiana.
[0028] At the same time, the spore count of the pure spore sample was performed using a blood cell counting plate (02270113, Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd.) to obtain the spore number. Figure 6 As shown in the figure, the results of RT-qPCR are consistent with the results of the blood cell counting plate. The standard curve was drawn with the CT value as the horizontal axis and the number of spores as the vertical axis. The standard curves of the pure spore samples taken after 48h, 60h, 96h, 168h, and 216h of culture were as follows: Figure 7 The linear regression equation corresponding to the standard curve of the pure spore sample taken after 48 hours of culture is: y = -559968x + 10 7 (R 2 =0.9305), the linear regression equation of the standard curve of the pure spore sample taken after 60h of culture is: y = -5*10 6 x+2*10 8 (R 2 =0.9642), the linear regression equation corresponding to the standard curve of the pure spore sample taken after 96h of culture is: y = -10 7 x+4*10 8 (R 2 =0.8911), the linear regression equation of the standard curve of the pure spore sample taken after 168h of culture is: y = -3*10 8 +9*10 9 (R 2 =0.7917), the linear regression equation of the standard curve of the pure spore sample taken after 216h of culture is: y = -6*10 8 x+2*10 10 (R 2 =0.9636).
[0029] S3: Take 200g of peeled potatoes, add 1000ml of water, boil for 30min, filter with gauze, then weigh 20g of glucose and 20g of agar powder and add them to the filtrate, distilled water to 1L, sterilize at 121℃ for 30min to obtain PDA solid culture medium; inoculate the strain of Beauveria bassiana BbFZ-51 on PDA solid culture medium under sterile conditions, culture at 28℃ for 12h, 24h, 30h, 48h, 60h, and 96h before sampling. The sample is a mixture of mycelium and spores, which is quick-frozen in liquid nitrogen and stored in a -80℃ refrigerator. The total RNA of the sample is extracted using an RNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd.), and reverse transcribed using oligo(dT) primers to obtain the corresponding cDNA as a template, and RT-qPCR is performed using primers A1176-F / A1176-R and A5201-F / A5201-R, respectively. The Bb-actin gene was selected as the internal reference. The internal reference primers were published in [CHO EM, KIRKLAND BH, HOLDER DJ, et al. Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana [J]. Microbiology, 2007, 153 (10): 3438–3447.], and the sequences were: F: 5'-TTGGTGCGAAACTTCAGCGTCTAGTC-3', R: 5'-TCCAGCAAATGTGGATCTCCAAGCAG-3'. The RT-qPCR reaction system included: 1 μl of cDNA template, 1 μl of 10 μM upstream primer, 1 μl of 10 μM downstream primer, and Hieff TMqPCR SYBR Green Master Mix (Low Rox Plus) (Yisheng Biotechnology (Shanghai) Co., Ltd.) 25μl, sterile ultrapure water 22μl. RT-qPCR reaction conditions are: 94℃ pre-denaturation for 2min; 94℃ denaturation for 15s, 60℃ annealing / extension for 30s, 35 cycles; melting curve analysis conditions are: 95℃ 15s, 60℃ 1min, 95℃ 15s. After the RT-qPCR experiment, the CT value obtained by primers A5201-F / A5201-R was substituted into the standard curve established for spores at each sampling time point in S2 to calculate the absolute content of Beauveria bassiana BbFZ-51 spores in samples at different sampling time points (48h, 60h, 96h), and further calculated the relative content. The calculation formula was: (absolute content of spores at a certain sampling time point / absolute content of spores at a sampling time point of 48 hours) × 0.1; the CT value obtained by primers A1176-F / A1176-R was used to calculate the relative content of Beauveria bassiana BbFZ-51 hyphae in samples at different sampling time points (12h, 24h, 30h, 48h, 60h, 96h), and the calculation formula was: (CT value at a certain sampling time point / CT value at a sampling time point of 12 hours) × 0.1. The relative content change curve was drawn as shown in the figure. Figure 8 It should be noted that the multiplication by 0.1 is to make the numerical value more beautiful and clear when drawing. It will not affect the proportional relationship between the data. The original value is scaled in this way mainly because the absolute content value or CT value directly obtained may be too large, and it may not be intuitive enough to use it directly for drawing.
[0030] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A primer set for detecting spores and hyphae of Beauveria bassiana, characterized in that: The primer set includes primers for detecting spores of Beauveria bassiana and primers for detecting hyphae of Beauveria bassiana; The primers for detecting spores of Beauveria bassiana are primers for detecting the A5201 gene of Beauveria bassiana, and the nucleotide sequence of the A5201 gene is shown in SEQ ID NO.7; the primers for detecting mycelium of Beauveria bassiana are primers for detecting the A1176 gene of Beauveria bassiana, and the nucleotide sequence of the A1176 gene is shown in SEQ ID NO.
1.
2. The primer set according to claim 1, characterized in that: The nucleotide sequences of the primers for detecting the Beauveria bassiana A5201 gene are: upstream primer 5′-ATGAGCACTTTGAGCTTCGAC-3′, downstream primer 5′-TTATCTTCTCCGTGGGCTGTC-3′; the nucleotide sequences of the primers for detecting the Beauveria bassiana A1176 gene are: upstream primer 5′-ATGAAGACTGCTGTTGGTATTGTTT-3′, downstream primer 5′-TCATATTTCTCGCGAGAAACGAC-3′.
3. Use of the primer set according to any one of claims 1 to 2 in detecting the transcription level of the A5201 gene and the A1176 gene of Beauveria bassiana, characterized in that: The application is for non-disease diagnosis and treatment purposes.
4. Use of the primer set according to any one of claims 1 to 2 in detecting spores and hyphae of Beauveria bassiana, characterized in that: The application is for non-disease diagnosis and treatment purposes.
5. A method for detecting the transcription level of Beauveria bassiana A5201 gene and A1176 gene by RT-qPCR, characterized in that: The method is for non-disease diagnosis and treatment purposes, and comprises the step of adding the primers described in claim 2 to the reaction system to perform single-plex real-time fluorescence quantitative PCR amplification.
6. The method according to claim 5, characterized in that: The reaction system of the single-plex real-time fluorescence quantitative PCR amplification is: 1 μl of cDNA template, 1 μl of upstream primer, 1 μl of downstream primer, 25 μl of Hieff™ qPCR SYBR Green Master Mix, and 22 μl of sterile ultrapure water.
7. The method according to claim 5, characterized in that: The reaction conditions for the single-plex real-time fluorescence quantitative PCR amplification were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing / extension at 60°C for 30 s, and 35 cycles; and the melting curve analysis conditions were: 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s.