A kit for identifying single and double nuclear mycelium of cultivated variety of pleurotus geesterum

Abstract

This invention discloses a kit for identifying monokaryotic and dikaryotic mycelial samples of the main cultivated varieties of *Pleurotus ostreatus*. It includes four pairs of molecularly characteristic fluorescent SSR marker primers. This molecular identification technique can exclude dikaryotic samples with the A / a genotype from a large number of *Pleurotus ostreatus* single-spore isolated populations, accurately and efficiently identifying monokaryotic (A or a genotype) or self-crossed dikaryotic (A / A or a / a genotype) mycelial samples. It increases the accuracy of traditional microscopic methods for identifying monokaryotic mycelia from only 70% to over 95%, and is faster and more efficient.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a kit for identifying monokaryotic and dikaryotic mycelial samples of the main cultivated varieties of Pleurotus ostreatus. Background Technology

[0002] Oyster mushroom ( Pleurotus pulmonarius Oyster mushroom (Pleurotus ostreatus), originally from India and also known as Indian abalone mushroom, belongs to the class Basidiomycetes, order Agaricales, family Pleurotaceae, and genus Pleurotus. It is a lung-type Pleurotus within the oyster mushroom family. Originally a wild edible fungus found in tropical and subtropical regions, it has been domesticated and bred for artificial cultivation. With the rapid development of the oyster mushroom industry, cultivation methods are constantly being updated and improved, and oyster mushroom cultivation is gradually shifting from facility-based cultivation to factory-style production. However, suitable oyster mushroom varieties for factory production have not been updated in a timely and effective manner. Oyster mushroom producers have an increasing demand for new varieties that are high-quality, high-yielding, stress-resistant, and disease-resistant. Therefore, oyster mushroom breeding is currently very important and has become a key research direction for many research institutes and enterprises.

[0003] Currently, research on oyster mushroom breeding is relatively weak, primarily due to a lack of efficient breeding techniques. Hybrid breeding is the most common and effective technique for new variety selection. It typically involves selecting appropriate parents and obtaining corresponding mononuclear materials. Numerous hybrids are then obtained through appropriate hybridization methods. After identification, these hybrids undergo initial screening via mycelial culture and fruiting trials, eliminating many hybrids with mediocre or poorer traits than their parents, retaining only a few superior hybrids for further screening. Finally, the superior hybrids obtained from the second screening are subjected to large-scale demonstration and promotion trials to obtain superior oyster mushroom hybrid varieties with stable and excellent traits. Oyster mushroom hybrid breeding mainly includes mononuclear hybridization, single-double hybridization, and protoplast fusion breeding. Among these, mononuclear hybridization produces hybrids with relatively uniform traits, better integrating two or more superior traits within a species into a new variety, often exhibiting strong viability and hybrid vigor, hence its widespread application in oyster mushroom breeding.

[0004] For heterothallic hybridization of *Pleurotus ostreatus*, the first step is to isolate haploid (monokinetic) hyphae from diploid (binukid) strains of the two parent strains. These haploid hyphae are then hybridized to form a combination of binucleate mycelia with clamp connections, which is essential for normal fruiting. Therefore, single-spore isolation is a crucial step in monokaryotic hybridization breeding. The commonly used method involves diluting the spore solution and then observing the hyphae under a microscope to determine if they are monokaryotic. This method is very time-consuming and labor-intensive, and often lacks clarity under the naked eye, leading to misidentification—specifically, mistaking binucleate hyphae for monokaryotic hyphae—thus increasing the failure rate of subsequent monokaryotic hybridization breeding.

[0005] Simple repeat sequences (SSRs), also known as microsatellite DNA, are codominant molecular markers that are reliable, stable, and easy to operate, making them suitable for high-throughput applications and thus widely used in marker-assisted breeding. Using codominant fluorescent SSR labeling technology, heterozygous SSR loci can be screened for in diploid varieties of *Pleurotus ostreatus*. These SSR loci can then be used to distinguish between monokaryotic and dikaryotic mycelial samples. Specifically, dikaryotic samples have two different alleles, A and a, at this locus, resulting in a diploid A / a genotype, which appears as a double peak in capillary electrophoresis. In contrast, monokaryotic samples have only one allele, either A or a, at this locus, resulting in either a genotype, which appears as a single peak in capillary electrophoresis. It should be noted that during the isolation of single spores, self-pollinating binucleate hyphae may also occur at a very low frequency. This is when mononuclear spores of the same genotype mate with each other to form homozygous diploid A / A or a / a type binucleate hyphae. These hyphae will also appear as a single peak on the capillary electrophoresis peak pattern. Therefore, the heterozygous SSR locus cannot distinguish between self-pollinating binucleate hyphae and mononucleate hyphae. Summary of the Invention

[0006] The purpose of this invention is to provide a method for identifying the efficiency of monokaryotic and binkaryotic mycelia of Pleurotus ostreatus based on molecular characteristic SSR marker primers, and also relates to the application of said primers in the identification of monokaryotic and binkaryotic fungal samples.

[0007] The technical solution adopted in this invention is:

[0008] The molecularly characteristic fluorescent SSR marker primer pair used to identify mono- and dikaryotic mycelial varieties of the main cultivated oyster mushroom species is any one of the following pairs:

[0009] (1) Primer Pg_U4798:

[0010] Upstream primer Pg_U4798F: 5′-CAAGTGACTCCGAACCCAAT-3′;

[0011] Downstream primer Pg_U4798R: 5′-CTACCGAGCAACTCCTCGAC-3′;

[0012] (2) Primer Pg_U2510:

[0013] Upstream primer Pg_U2510F: 5′-CTTGTCTTGCTGCACCCAC-3′;

[0014] Downstream primer Pg_U2510R: 5′-GACTCTGGGACTTGGGATGA-3′;

[0015] (3) Primer Pg_U2278:

[0016] Upstream primer Pg_U2278F: 5′-CACAGTGAAGAGGGGGACAT-3′;

[0017] Downstream primer Pg_U2278R: 5'-CTTATGCTGCTTTGACAGCG-3';

[0018] (4) Primer Pg_U6702:

[0019] Upstream primer Pg_U6702F: 5′-CGTTGAGTCGAAACGTGAGA-3′;

[0020] Downstream primer Pg_U6702R: 5′-TCTCTTTGACTCGCTCCCTC-3′;

[0021] In each molecule's characteristic fluorescent SSR-labeled primer pair, one primer has a fluorescent group attached to its 5' end.

[0022] Preferably, the fluorescent group of the upstream primer is FAM, and the fluorescent group is attached to the 5' end of the upstream primer.

[0023] This invention further provides the application of the molecularly characteristic fluorescent SSR marker primer pair in identifying mono- and binkaloid mycelial samples of the main cultivated varieties of Pleurotus ostreatus.

[0024] The present invention also provides a kit for identifying monokaryotic and dikaryotic mycelial samples of the main cultivated varieties of Pleurotus ostreatus, comprising the molecularly characteristic fluorescent SSR labeled primer pair.

[0025] Preferably, the kit further includes an enzyme for PCR amplification.

[0026] This invention also provides a method for identifying monokaryotic and dikaryotic mycelial samples of the main cultivated varieties of Pleurotus ostreatus, comprising the following steps:

[0027] (1) Extract DNA from the mycelium sample of the main cultivated variety of Pleurotus ostreatus to be identified and add it to the PCR amplification system as an amplification template, or directly add the lysate of the mycelium sample of the main cultivated variety of Pleurotus ostreatus to be identified to the PCR amplification system.

[0028] (2) Then, the characteristic fluorescent SSR marker primer pair of the aforementioned molecules was added for PCR amplification.

[0029] (3) Capillary electrophoresis was used to detect the amplified products. If the detection result was a single peak, the sample of the main cultivated variety of Pleurotus ostreatus to be identified was a monokaryotic sample; if the detection result was a double peak, the sample of the main cultivated variety of Pleurotus ostreatus to be identified was a binucleate sample.

[0030] Preferably, the PCR amplification system is: 2×T5 Super PCR Mix PAGE 10 L, 1.5g each of upstream and downstream primers. L, DNA template 3 L, add ddH2O to bring the total to 20. L.

[0031] Preferably, the PCR reaction conditions are: 98℃ / 2 min; 98℃ / 10 s, annealing at 53.8℃ for 10 s, 72℃ / 10 s, for 30 cycles; finally, the reaction is terminated at 72℃ / 2 min and 4℃.

[0032] Preferably, for capillary electrophoresis detection, 10 ml of Hi-Di and 80 μL of GeneScan™-500 LIZ Size Standard are mixed, centrifuged, and analyzed at 10 μL per well. L was dispensed into a 96-well internal standard plate and centrifuged;

[0033] The PCR amplification products were subjected to capillary electrophoresis, diluted according to the electrophoresis gel image, and centrifuged.

[0034] Add 0.5 μL of the diluted product to the prepared internal standard plate, mix well, centrifuge, place in a PCR instrument, and denature at 96℃ for 5 min.

[0035] Freeze rapidly at 20°C for 2 minutes, then centrifuge;

[0036] Insert into a DNA analyzer for capillary electrophoresis detection;

[0037] Data was collected using Data Collection 3.0 software.

[0038] The molecularly characteristic primers Pg_U4798, Pg_U2510, Pg_U2278, and Pg_U6702 of this invention are based on fluorescent SSR labeling technology. They were obtained after screening a large number of SSR primers developed based on transcriptome sequencing data among the main cultivated varieties of *Pleurotus ostreatus*. These primers exhibit high amplification efficiency and good stability, accurately identifying whether *Pleurotus ostreatus* samples are monokaryotic or multikaltic. Using these primers, a single peak indicates that the isolated sample is a monokaryotic *Pleurotus ostreatus* sample, while a double peak indicates that the isolated sample is a dikaryotic *Pleurotus ostreatus* sample. Using primers modified with the fluorescent group FAM, 45 *Pleurotus ostreatus* isolated samples were detected. All four primer pairs detected monokaryotic samples, with a detection rate as high as 90%. Therefore, Pg_U4798, Pg_U2510, Pg_U2278, and Pg_U6702 are molecularly characteristic primers for rapid identification of monokaryotic and dikaryotic *Pleurotus ostreatus* samples.

[0039] Currently, there are no reports on the rapid identification of monokaryotic and dikaryotic parents of *Pleurotus ostreatus* using molecularly characteristic fluorescent SSR markers. However, the molecular identification technology of this invention can exclude dikaryotic samples with A / a genotypes from a large number of *Pleurotus ostreatus* single-spore isolated populations, accurately and efficiently identifying monokaryotic (A or a genotype) or self-crossed dikaryotic (A / A or a / a genotype) mycelial samples. This increases the accuracy of traditional microscopic examination methods for identifying monokaryotic organisms from only 70% to over 95%, and is faster and more efficient. Attached Figure Description

[0040] Figure 1 The image shows the genotype results of amplification of Hangzhou No. 2 mononuclear and binucleate hyphae using primer U4798. From top to bottom, the images show binucleate bimodal and two types of mononucleate unimodal hyphae.

[0041] Figure 2 The image shows the genotype results of amplification of Taixiu 57 mononuclear and binucleate hyphae using primer U2510. From top to bottom, the images show binucleate bimodal and two types of mononucleate unimodal hyphae.

[0042] Figure 3 The image shows the genotype results of amplifying mono- and binucleate mycelia of Pleurotus ostreatus using primer U2278. From top to bottom, the images show binucleate bimodal and two types of mononucleate unimodal mycelia.

[0043] Figure 4 The image shows the genotypes of Jinxiu mono- and binucleate hyphae amplified using primers U6702. From top to bottom, they represent binucleate bimodal and two types of mononucleate unimodal hyphae. Detailed Implementation

[0044] Example 1

[0045] SSR primer design.

[0046] The development of primers for differentiating monokaryotic and dikaryotic mycelia of Pleurotus ostreatus consists of three steps.

[0047] First, transcriptome sequencing was performed on the oyster mushroom variety Hangxiu 2. Fifty pairs of SSR markers were screened from the assembled Unigene data, and their effectiveness was verified using PCR technology.

[0048] Second, fluorescent SSR primers were used to label 41 effective SSR markers, and 19 SSR heterozygous sites were screened by capillary electrophoresis of PCR products.

[0049] Third, the heterozygosity of the 19 SSR heterozygous loci in five main cultivated varieties of *Pleurotus ostreatus* (Hangxiu No. 1, Nongxiu No. 1, Taixiu 57, Jinxiu, and Xiuzhengu 18) was screened, and four SSR heterozygous loci were obtained, meaning they were heterozygous in all five varieties. Therefore, the four SSR markers developed in this technology can be used to identify monokaryotic and dikaryotic mycelia in all main cultivated varieties of *Pleurotus ostreatus*. Table 1 shows the relationships between the four SSR markers and the designed primers for amplification, and Table 2 shows the primer amplification fragments.

[0050] Table 1 SSR markers for main cultivated varieties

[0051]

[0052] Table 2 Primer Amplification Fragments

[0053]

[0054] Example 2

[0055] Four pairs of molecularly characteristic fluorescent SSR marker primers designed in Example 1 were used to screen and identify mono- and binkaloid mycelial samples of the main cultivated varieties of Pleurotus ostreatus.

[0056] 1. Main reagents and instruments.

[0057] The main reagents included a plant genomic DNA extraction kit (BioTeke, Beijing) and a 2×T5 Super PCR Mix (PAGE) (TSINGKE, Beijing). The fluorescent dye added to the SSR forward primers was FAM (blue), and the SSR primers were synthesized by Beijing TSINGKE Biotechnology Co., Ltd. (Beijing). The internal standard reagent used for capillary electrophoresis detection was Hi-Di. TM Formamide (Applied Biosystems), GeneScan TM -500 LIZ Size Standard (AppliedBiosystems).

[0058] The main instruments include a PCR instrument (Life ECO, Bioer, Hangzhou), a DNA analyzer (96-well plate, ABI3730 XL Genetic Analyzer, Applied Biosystems, USA), and a NanoDrop 2000 (ThermoScientific, USA).

[0059] 2. Genomic DNA extraction.

[0060] Genomic DNA was extracted from the mycelia of five main cultivated varieties of *Pleurotus ostreatus* (Hangxiu No. 1, Nongxiu No. 1, Taixiu 57, Jinxiu, and Xiuzhengu 18) after freeze-crushing. The DNA extraction method followed the instructions of a plant genomic DNA extraction kit. The concentration of the extracted DNA was determined, and the results were detected by agarose gel electrophoresis. The extracted DNA was stored at -20℃ for later use.

[0061] 3. SSR-PCR analysis.

[0062] SSR markers were derived from previous screening of transcriptome sequencing data of *Pleurotus ostreatus* var. *hangxiu*.

[0063] To screen 20 polymorphic SSR markers The L SSR-PCR amplification system consisted of: 2×T5 Super PCR Mix (PAGE) 10 L, FAM fluorescently labeled SSR forward and reverse primers (10 mol·L⁻¹) -1 1.5 each L, DNA template (20 ng·L) -1 3 L, add ddH2O to bring the total to 20. L.

[0064] The PCR reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, annealing for 10 s, 72℃ for 10 s, for 30 cycles; and finally 72℃ for 2 min, and terminated at 4℃.

[0065] Annealing temperatures for different SSR markers were screened and optimized from 50℃ to 60℃, and the final annealing temperature was determined to be 53.8℃. PCR amplification products were first detected by 1.5% agarose gel electrophoresis, and usable SSR markers were then detected by capillary electrophoresis to analyze their polymorphism among the main cultivated varieties of Pleurotus ostreatus.

[0066] 4. Capillary electrophoresis detection.

[0067] Mix 10 ml Hi-Di and 80 μL GeneScan™-500 LIZ Size Standard, centrifuge, and divide into 10 samples per well. Aliquots were distributed into 96-well internal standard plates and centrifuged. SSR-PCR products were electrophoresed, and diluted according to the gel image (minimum detectable standard: 0.1 ng / L). -1 Centrifuge. Add 0.5 μL of the diluted product to the allocated internal standard plate, mix well, centrifuge, and place in a PCR instrument for denaturation at 96℃ for 5 min. Quickly freeze at 20℃ for 2 min, then centrifuge. Perform capillary electrophoresis in a DNA analyzer. Collect data using Data Collection 3.0 software.

[0068] 5. Data analysis.

[0069] The raw data collected by Data Collection 3.0 software was analyzed using GeneMapper 4.1 software. The software system directly provides the accurate value (bp) of the target SSR fragment by comparing the position of the target peak with the internal standard GeneScanTM-500 LIZ Size Standard in the same lane. Allelic data for heterozygous sites are recorded as X / Y, where X and Y represent the numerical values ​​of the two different allelic variants at that site.

[0070] 6. The results of the identification.

[0071] 6.1 Dual-core results

[0072] The genotypes of Pg_U4798, Pg_U2510, Pg_U2278 and Pg_U6702 markers of five main cultivated varieties of Pleurotus ostreatus were detected, and the results are shown in Table 3.

[0073] Table 3. Genotypes of dikaryotic mycelia of different varieties of Pleurotus ostreatus

[0074]

[0075] The results showed that the molecular characteristic primers Pg_U4798, Pg_U2510, Pg_U2278 and Pg_U6702 could detect four genotypes (207 / 213, 274 / 295, 146 / 152 and 261 / 267) in the binucleate hyphae of the five varieties.

[0076] 6.2 Single-core results

[0077] Amplification of the five monokaryotic or self-crossed dikaryotic hyphae showed a single peak, with genotypes of 207 / 207, 213 / 213, 274 / 274, 295 / 295, 146 / 146, 152 / 152, 261 / 261, or 267 / 267. Therefore, the peak patterns can be used to identify monokaryotic hyphae (including self-crossed dikaryotic hyphae). The results are shown in Table 4.

[0078] Table 4. Genotypes of monokaryotic mycelia of different varieties of Pleurotus ostreatus

[0079]

[0080] This indicates that genotypic combination differences based on fluorescent SSR marker technology can serve as SSR fingerprints for monokaryotic and dikaryotic strains of *Pleurotus ostreatus*. In future *Pleurotus ostreatus* breeding work, we will first isolate monokaryotic mycelial samples from dikaryotic mycelial samples, and then use SSR fingerprints to identify monokaryotic and dikaryotic mycelia. Figure 1As shown, under primer U4798, the binucleate mycelial samples of the main cultivated *Pleurotus ostreatus* strain Hangxiu 2 showed two peaks at 207 and 213, while the monokaryotic mycelial samples of Hangxiu 2 only showed one peak between 207 and 213. The presence of a single peak indicates successful isolation of a single spore, which can be used as material for subsequent hybridization breeding; the presence of two peaks indicates failed isolation of a single spore. Under the same principle, Figure 2 , Figure 3 and Figure 4 The results showed that using primers U2510, U2278, and U6702 to amplify samples of Taixiu 57, Xiuzhengu 18, and Jinxiu yielded the same result: two peaks were observed in binucleate mycelial samples, while a single peak was observed in mononucleate mycelial samples. This indicates that the genotype combinations of fluorescent SSR markers are significantly different. This method provides an efficient and convenient molecular technique for the identification of mononucleate and binucleate mycelial materials in Xiuzhengu breeding materials.

[0081] Example 3

[0082] One hundred samples of the Hangxiu No. 2 single-spore strain were used as test strains, and their verification was carried out using two methods: conventional microscopic examination and the molecular characteristic fluorescent label U2510 of this application.

[0083] Traditional microscopic examination identifies monokaryotic hyphae as those without clamping junctions and dikaryotic hyphae as those with clamping junctions. Molecular fluorescence U2510 labeling identifies monokaryotic hyphae as those with a single peak at 274 / 274 or 295 / 295, and dikaryotic hyphae as those with a double peak at 274 / 295. Since monokaryotic hyphae do not produce fruiting bodies after cultivation (no fruiting), while dikaryotic hyphae will produce fruiting bodies (fruiting), fruiting tests are used as the criterion to verify the correctness of both methods.

[0084] Table 5 Comparison of detection rates of monokaryotic hyphae by traditional microscopy and molecular markers

[0085]

[0086] The results are shown in Table 5. The experimental results indicate that, using traditional microscopy, 20 samples of monokaryotic hyphae were identified as dikaryotic hyphae, while molecular fluorescent labeling identified 28 dikaryotic hyphae. Finally, in a fruiting experiment, 29 out of the 100 samples formed fruiting bodies. Therefore, the accuracy rate of microscopic examination was 69%, while that of molecular labeling was 96%, representing a significant improvement in accuracy. Furthermore, microscopic examination is time-consuming, with a typical daily examination capacity of approximately 50 samples per person. Using molecular labeling, a single person can process 500 samples per day, significantly increasing the speed.

Claims

1. A pair of molecularly characteristic fluorescent SSR marker primers for identifying mono- and dikaryotic mycelial samples of the main cultivated varieties of Pleurotus ostreatus, characterized in that, For any of the following pairs: (1) Primer Pg_U4798: Upstream primer Pg_U4798F: 5′-CAAGTGACTCCGAACCCAAT-3′; Downstream primer Pg_U4798R: 5′-CTACCGAGCAACTCCTCGAC-3′; (2) Primer Pg_U2510: Upstream primer Pg_U2510F: 5′-CTTGTCTTGCTGCACCCAC-3′; Downstream primer Pg_U2510R: 5′-GACTCTGGGACTTGGGATGA-3′; (3) Primer Pg_U2278: Upstream primer Pg_U2278F: 5′-CACAGTGAAGAGGGGGACAT-3′; Downstream primer Pg_U2278R: 5'-CTTATGCTGCTTTGACAGCG-3'; (4) Primer Pg_U6702: Upstream primer Pg_U6702F: 5′-CGTTGAGTCGAAACGTGAGA-3′; Downstream primer Pg_U6702R: 5′-TCTCTTTGACTCGCTCCCTC-3′; In each molecule's characteristic fluorescent SSR-labeled primer pair, one primer has a fluorescent group attached to its 5' end.

2. The molecularly characteristic fluorescent SSR-labeled primer pair according to claim 1, characterized in that, The fluorescent group of the upstream primer is FAM, and the fluorescent group is attached to the 5' end of the upstream primer.

3. The application of the molecularly characteristic fluorescent SSR-labeled primer pair according to claim 1 or 2 in identifying mono- and dikaryotic samples of the main cultivated varieties of Pleurotus ostreatus, characterized in that, The main cultivated varieties of oyster mushroom are at least one of Hangxiu No. 1, Nongxiu No. 1, Taixiu 57, Jinxiu and Xiuzhengu 18.

4. A kit for identifying monokaryotic and dikaryotic mycelial samples of the main cultivated varieties of Pleurotus ostreatus, characterized in that, Includes the molecularly characteristic fluorescent SSR-labeled primer pair as described in claim 1 or 2.

5. The reagent kit according to claim 4, characterized in that, It also includes enzymes used for PCR amplification.

6. A method for identifying monokaryotic and dikaryotic mycelial samples of main cultivated varieties of Pleurotus ostreatus, characterized in that, Includes the following steps: (1) Extract DNA from the mycelium sample of the main cultivated variety of Pleurotus ostreatus to be identified and add it to the PCR amplification system as an amplification template, or directly add the lysate of the mycelium sample of the main cultivated variety of Pleurotus ostreatus to be identified to the PCR amplification system. (2) Then, add the molecularly characteristic fluorescent SSR marker primer pair described in claim 1 or 2 for PCR amplification. (3) Capillary electrophoresis was used to detect the amplification products. If the result was a single peak, the sample of the main cultivated variety of *Pleurotus ostreatus* to be identified was a monokaryotic sample; if the result was a double peak, the sample of the main cultivated variety of *Pleurotus ostreatus* to be identified was a dikaryotic sample. The main cultivated varieties of oyster mushroom are at least one of Hangxiu No. 1, Nongxiu No. 1, Taixiu 57, Jinxiu and Xiuzhengu 18.

7. The method according to claim 6, characterized in that, The PCR amplification system was: 2×T5 Super PCR Mix PAGE 10 L, 1.5g each of upstream and downstream primers. L, DNA template 3 L, add ddH2O to bring the total to 20. L.

8. The method according to claim 6, characterized in that, The PCR reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, annealing at 53.8℃ for 10 s, and 72℃ for 10 s, for 30 cycles; finally, the reaction was terminated at 72℃ for 2 min and 4℃.

9. The method according to claim 6, characterized in that, For capillary electrophoresis, mix 10 ml of Hi-Di and 80 μL of GeneScan™-500 LIZ Size Standard, centrifuge, and perform 10 saturations per well. L was dispensed into a 96-well internal standard plate and centrifuged; The PCR amplification products were subjected to capillary electrophoresis, diluted according to the electrophoresis gel image, and centrifuged. Add 0.5 μL of the diluted product to the prepared internal standard plate, mix well, centrifuge, place in a PCR instrument, and denature at 96℃ for 5 min. Freeze rapidly at 20°C for 2 minutes, then centrifuge; Insert into a DNA analyzer for capillary electrophoresis detection; Data was collected using Data Collection 3.0 software.

Images