Abstract
Boletus reticulatus is a wild edible fungus with high nutritional value in Yunnan Province. In this study, B. reticulatus was used as the research object to diagnose the species characteristics. A commercial kit was used to extract the DNA of various fungi, and the quality of DNA was determined by using universal fungus primers. Through sequence alignment, the spt5 gene was selected as the species-specific gene of B. reticulatus. This gene was then qualitatively and quantitatively analyzed by PCR. In the qualitative detection, the spt5 amplification products were only found in B. reticulatus which proved its good specificity. Meanwhile, SYBR Green I based quantitative PCR results were highly sensitive, and the limit of detection was 0.04 ng of genomic DNA. These experiments illustrated that spt5 is an ideal species-specific gene for the quantitative and qualitative detection of B. reticulatus. This method is also suitable for the analysis of the processed samples of B. reticulatus and the determination of the adulteration of edible wild mushrooms.
1. Introduction
The fruiting body of Boletus reticulatus is thick and delicate [1], and the nutritional value is extremely rich [2, 3]. B. reticulatus is not only edible but also has certain health effects [4], such as the treatment of numbness of the hands and feet [5], lumbago and leg pain, and infertility [6]. In the market, the price of B. reticulatus is more expensive than that of other fungi, food adulteration may occur, and food poisoning may even be reported in severe cases. Therefore, B. reticulatus should be identified to avoid this phenomenon. To improve the current situation of adulteration and protect the rights and interests of consumers, an efficient and rapid wild edible fungus detection method should be established.
Traditional detection methods include morphological identification, zoological detection, and physical and chemical detection. Morphological identification is based on the determination of the shape, odor, color, and secretions of fruiting bodies [7]. However, many types of wild fungi exist in Yunnan, and many wild fungi are highly similar in shape and cannot be directly distinguished [8]. Some deep-processed products can also severely damage the morphology of wild fungi, so morphological identification has certain limitations. Zoological detection refers to the use of animals to test whether edible fungi are toxic, which is not representative. The physical and chemical techniques are based on the analysis of unique markers in food to achieve product identification, and it is widely used in the field of food detection. With the continuous development and application of various technologies such as chromatography, mass spectrometry, and spectroscopy, chemical methods have been rapidly developed and perfected, playing an active and effective role in monitoring poor manufacturers [9–11]. However, with the advancement of science and technology, food adulteration technology is becoming increasingly sophisticated [12, 13]. By artificially adding chemical components, ordinary physical and chemical detection methods cannot easily identify the authenticity of food ingredients [14].
With the rapid development of modern biotechnology, molecular biological detection has been widely used to identify food sources [15, 16]. DNA-based forgery detection mainly relies on polymerase chain reaction (PCR) to amplify highly specific DNA fragments in the genome to achieve detection purposes [17, 18]. The techniques that fall under DNA-based forgery detection mainly include single-strand conformation polymorphism technology (SSCP), random amplified polymorphic DNA technology (RAPD), simple sequence repeat technology (SSR), intersimple sequence repeat technology (ISSR), and DNA amplification fingerprinting technology [19–21]. However, these technologies either need to design multiple pairs of primers and amplify multiple targets in the early stage or sequence the amplified products in the later stage. The purpose of detection can be achieved by sequence comparison, which takes a long time. Compared with the above techniques, detection technology based on amplifying species-specific gene (also called endogenous reference gene) is more convenient, specific, and economical [22].
Normal qualitative PCR has the advantages of low-cost, convenience, and wide applicability. Real-time fluorescent quantitative PCR technology has the advantages of high sensitivity, strong specificity and accuracy, short detection time, data visualization, and high automation. It is widely used in the fields of genetic modification detection, clinical medicine, and research and development of new disease-resistant crops. This study mainly used normal PCR and real-time fluorescent quantitative PCR technology to achieve qualitative and quantitative detection of species-specific gene of spt5 in B. reticulatus.
In this study, the DNA of various edible fungi was extracted using a commercial kit, and the DNA quality was determined by PCR using universal fungus primers. Through multiple sequence alignment, the spt5 gene is selected as the species-specific gene of B. reticulatus. This gene was then qualitatively and quantitatively analyzed by PCR using substrate genomic DNA of B. reticulatus and other species. The minimum detection limit was 0.04 ng of genomic DNA by SYBR Green fluorescence quantitative PCR, and the spt5 amplification products were not found in the 10 other species. Allelic variation was not detected in B. reticulatus. These experiments proved that spt5 is an excellent species-specific gene for the quantitative and qualitative detection of B. reticulatus. It can be used to analyze B. reticulatus and its processed samples and determine the adulteration of wild mushrooms.
2. Materials and Methods
2.1. Materials and Reagents
This study used 11 species of fungus samples, such as Boletus reticulatus, Boletus magnificus W.F.Chiu, Lentinus edodes, Agrocybe aegirit, Leccinum extremiorientale (L.Vass.) Singer, Boletus griseus Frost, Boletus sinicus W.F.Chiu, Russula virescens, Collybia albuminosa, Lactarius volemus, and Pleurotus eryngii. These samples were purchased from local markets in Yunnan Province, China.
The following reagents were used in the study: Ezup column fungal genomic DNA extraction kit purchased from Sangon Biotech (Shanghai) Co., Ltd.; 50× TAE buffer; EDTA (pH 8.0); 10 mg/mL RNase A, 20 mg/mL proteinase K, ethidium bromide (10 mg/mL), and DNA marker DL 2000 purchased from Tiangen Biotech (Beijing) Co., Ltd.; and 6× loading buffer purchased from Takara Biomedical Technology (Beijing) Co., Ltd. The primers used in the PCR reaction were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
2.2. DNA Extraction and Purification
About 50–100 mg of each fresh fungus was ground into powder with liquid nitrogen and transferred into a 1.5 mL centrifuge tube. The steps in the kit were followed to perform the experiment. The purity and concentration of the extracted DNA were measured by using the microconcentration analyzer NanoDrop 2000. Each sample was measured three times. Finally, the extracted DNA was placed at −20°C till used for the subsequent experiments.
2.3. Qualitative Detection of the Extracted Genomic DNA Using ITS Universal Fungal Primer
To verify whether the extracted DNA can be used for PCR amplification, the universal primers of fungal internal transcribed spacer (ITS) rDNA (Table 1) were used to amplify the genomic DNA of the 11 samples.
PCR assays were conducted in a total volume of 25 μL on an ABI SimpliAmp thermal cycler (Applied Biosystems, USA) (Table 2). The PCR amplification conditions were as follows: predenaturation (95°C, 5 min); 30 cycles of denaturation (95°C, 30 s), annealing (58°C, 30 s), and extension (72°C, 30 s); and termination extension (72°C, 10 min). The 5 μL of each PCR product was then analyzed by 2% agarose gel electrophoresis (containing 0.1 g/mL EB) [23].
2.4. Screening of Species-Specific Gene in B. reticulatus
The gene information of B. reticulatus was searched in the nucleotide database of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and BLASTN alignment analysis was performed. The gene with low homologous sequences and high species specificity was selected as the candidate of endogenous reference gene.
Aiming at the sequence of candidate genes, Primer Premier 5.0 software (PREMIER Biosoft, San Francisco, USA) was used to design primers. BLASTN comparative analysis was performed on the designed primers to ensure the specificity, and the detailed sequences are given in Table 1.
2.5. Verification of the Specificity of the Selected Species-Specific Gene
The genomic DNA of the 11 mushroom species was used as the template to qualitatively verify the interspecies specificity of the species-specific gene in B. reticulatus. The PCR amplification system and conditions were the same with those of ITS amplification.
2.6. Detection Sensitivity of the Species-Specific Gene in B. reticulatus
The genomic DNA of B. reticulatus was selected as the target, and the sample was diluted tenfold in a gradient of 20, 2, 0.2, 0.02, 0.002, and 0.0002 ng/μL; ddH2O was used as a negative control. The qualitative and quantitative detection was performed to determine the detection sensitivity.
In the qualitative detection, the PCR conditions were the same with those of ITS amplification. The quantitative detection sensitivity of the species-specific gene was evaluated in a final volume of 25 μL. Each reaction mixture contained 1× SYBR Green Mix, 200 nm of each primer, and 2 μL of genomic DNA. Real-time PCR reactions were performed on an ABI Step One Plus detection system (Applied Biosystems, Foster City, USA) with the following program: 2 min at 50°C, 10 min at 95°C, 40 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 68°C. Then, melting curve analysis was performed as follows: 15 s at 95°C, 20 s at 60°C, and 95°C at a heating rate of 0.5°C/s. The specificity of the species-specific gene can be verified by checking whether the melting curve has a single peak at the appropriate Tm value. The samples of each biological replicate were quantified, and the process was replicated twice. A standard curve relating the DNA quantities to the detection thresholds (Ct) was constructed.
3. Results and Discussion
3.1. Qualitative Verification of the Extracted Genomic DNA
In this experiment, the genomic DNA of 11 species of wild mushrooms was extracted by using the kits and detected by electrophoresis in 1% agarose gel. The test results are shown in Figure 1(a); although there are no visible bands in some lanes, this does not mean that DNA has not been successfully extracted. The lack of bands in electrophoresis results may be due to the low DNA concentration. As long as the target genomic DNA can be successfully amplified by PCR using universal primers of fungal, the extracted DNA can be used for the subsequent experiments.
(a)
(b)
The purity of DNA extraction plays a prerequisite role for subsequent PCR reactions. Extracting high-quality DNA is a key step in molecular biology tests. NanoDrop 2000 was used to determine the concentration and purity of the genomic DNA of the 11 wild bacteria, and each sample was repeatedly measured three times. The test results are given in Table 3. In general, the OD260/280 values among 1.7–1.9 can be judged as DNA, but the small fragment of degraded DNA cannot be observed in the electrophoretic diagram, and the concentration can still be measured when analyzed the absorbance value. When the OD260/280 value is out of the range mentioned above, it may be generated by other substances, such as protein, RNA, or impurities. In this table, the OD260/280 of the DNA of these 11 wild bacteria was between 1.7 and 2.0, indicating that the purity of the DNA was relatively high.
The genomic DNA of the 11 species of wild fungi was amplified by PCR using universal primers of fungal ITS rDNA, and the amplification results were detected by 2% agarose gel electrophoresis. The test results are shown in Figure 1(b). The ITS rDNA was effectively amplified from the genomic DNA of the 11 wild fungi, showing electrophoretic bands different from the blank. The bands were clear and tidy, which meets the requirements of PCR experiments. The electrophoretic results of A. aegirit (lanes 7-8) were shallow but not absent.
3.2. Selection of Species-Specific Genes in B. reticulatus
A qualified species-specific gene should have a low and constant copy number because low-copy genes tend to be relatively conserved, and their mutation rates are low; meanwhile, it also should exhibit low heterogeneity within the same species. The nucleotide of B. reticulatus was searched in NCBI, and 209 results were produced; every DNA sequence was analyzed by BLAST alignment. After the analysis, the spt5 gene (accession number: LC084605.1, Boletus reticulatus spt5 genes for spt5 protein) was selected as the candidate of species-specific gene. As shown in Figure 2, the spt5 gene exhibits low homology, and no other DNA sequences were aligned.
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(b)
3.3. Species Specificity Verification of spt5 Gene in B. reticulatus
To verify the species specificity of the spt5 gene in B. reticulatus, the genomic DNA of 11 of mushrooms was used as samples, and the primer pair spt5-F/R was used for common PCR amplification detection. The PCR products were analyzed by 2% agarose gel electrophoresis (containing 0.1 μg/mL EB). The results are shown in Figure 3.
The spt5 gene was successfully amplified from the genomic DNA of B. reticulatus, and the band was obvious, while the other mushrooms did not obtain the amplified products.
3.4. Detection Sensitivity of spt5 Gene in B. reticulatus
3.4.1. Qualitative Detection Results of spt5 Gene in B. reticulatus
To obtain the qualitative detection limit of the spt5 gene of B. reticulatus, the genomic DNA of B. reticulatus was series diluted. The amplified products were analyzed by 2% agarose gel electrophoresis, and the result is shown in Figure 4.
When the DNA template contents of B. reticulatus were 40, 4, and 0.4 ng, a clear and specific band appeared on the electrophoresis map, and the brightness gradually decreased. When the template content decreased to 0.04 ng, no product was appeared, which indicated that the detection limit of qualitative PCR was 0.4 ng.
3.4.2. Quantitative Detection Results of spt5 Gene in B. reticulatus
The SYBR Green real-time PCR amplification curve and standard curve are shown in Figure 5. In Figure 5(a), the amplification curve still occurred when the template content was as low as 0.04 ng, and the Ct value was significantly lower than the negative control. Thus, the detection limit of SYBR Green quantitative PCR was 0.04 ng.
(a)
(b)
As shown in Figure 5(b), we could obtain the linear formula of the Ct value and the content of the DNA template: Y = −2.728X + 26.048 (The value range of X is: > 0.04), and the correlation coefficient R2 was 0.995.
4. Conclusion
In this study, we screened and verified the species-specific gene, spt5, of B. reticulatus, and the results showed that the spt5 gene exhibited excellent species specificity, which are suitable for practical use in the qualitative and quantitative monitoring of B. reticulatus component. This method showed good specificity and sensitivity, and limit of detection was 0.04 ng of genomic DNA by SYBR Green fluorescence quantitative PCR. The results showed that this method can be used to evaluate the processed products of B. reticulatus or other types of products containing small amounts of B. reticulatus, and the DNA quantities of it reached the detection thresholds.
Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.
Ethical Approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31801635) and the Yunnan Education Department Scientific Research Fund Project (2018JS020).