Identification of s9ap used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of Pleurotus eryngii

Pleurotus eryngii is a kind of edible fungi with good quality, and it is popular among consumers. At present, some adulterated edible fungi are available in the market. The rights and interests of consumers can be ensured by establishing a practical edible fungi detection system. Among the existing methods for detecting food adulteration, endogenous reference gene amplification is convenient and reliable. However, no ideal endogenous reference gene is available for P. eryngii. In this study, s9ap was screened as an endogenous reference gene through sequence alignment. Qualitative and quantitative PCR analysis of this gene was carried out in one P. eryngii variety and 18 other species. The detection limit of quantitative PCR was 400 pg, and no s9ap amplification products were detected in the 18 other species. This study confirmed that s9ap was an ideal endogenous reference gene for the detection of P. eryngii. This method was also suitable for processed food products.


Introduction
Pleurotus eryngii is an edible mushroom, which is widely cultivated throughout the world; it is also known as king trumpet mushroom or king oyster mushroom [1], which belongs to basidio mycotina, hymenomycetes, color of the fruiting bodies and the characteristics of spores. However, this method mainly involves a subjective judgment. Hence, it cannot ensure the accuracy of the detection results. Moreover, the morphological identification method only applies to the fruiting body of fresh mushrooms, it's not applicable for the identification of the processed products of edible fungi [11]. In addition to the morphological characteristics, the analysis of specific volatile compounds using electronic nose technology and gas chromatographymass spectrometry can also identify edible fungi [12,13], this method is sensitive and fast [14]. Tagkouli, D et al. [15] studied the volatile characteristics of P. eryngii and Pleurotus ostreatus strains, and the results showed that ketones, alcohols, and toluene were the main components that distinguish P. ostreatus, while aldehydes and fatty acid methyl esters were the main components that distinguish P. eryngii. Edible fungi contain a large amount of protein and polysaccharide, it can also be detected by spectroscopic, chromatographic and mass spectrometry techniques to analyze the unique compound profile of each edible fungi. El Karkouri et al. [16] first applied MALDI-TOF-MS technology to identify truffles, which can reliably identify species and detect fake products in the truffle markets. However, these methods rely on large instruments and require complex data processing, their application to various groups of mushrooms is rather limited [17].
Considering that the genetic material is stable, molecular methods can be used to identify edible fungi and their processed products. Molecular biology technology is widely used in species identification [18], it has the advantage of fast, specific, sensitive, and accurate [19]. The commonly used molecular markers include simple sequence repeats (SSR), inter SSR (ISSR), random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), and sequence characterized amplified regions. These molecular markers are highly polymorphic and have been used in the identification of edible fungi species [20]. ISSR [21] and RAPD [22] have been used to identify P. eryngii. Other molecular biotechnologies include genome sequencing, which identifies edible fungi by sequencing specific DNA fragments. The commonly used gene fragments for classification and identification of fungi include: ribosome internal transcribed spacer (ITS), ribosome large subunit, RNA polymerase II (RNA polymerase II, RPB2), β-micro tube protein (β-tubulin), and elongation factor 1-α (EF1-α) [20]. Xiao-lan et al. [23] analyzed the ITS, EF1α, and RPB2 sequences of Bailinggu, P. eryngii, and P. nebrodensis. These three DNA fragment sequences can be used to distinguish three different species of edible fungi. With the development of rapid detection technology, isothermal amplification technology has also been used in the identification of edible fungi [24,25]. Endogenous reference gene analysis is broadly applied in food component source authentication and for the qualitative and quantitative evaluation of food samples. Endogenous reference gene is a conserved DNA sequence with species specificity, constant copy number and no allelic variation [26]. This method is convenient and economical, and it is a reliable and rapid choice for identifying wild species [27]. At present, the development and research of the endogenous reference genes are mainly focused on crops [28]. In recent years, it has been developed for the detection of mushrooms. It has been used to detect Tricholoma matsutake [11], P. ostreatus [27], and Flammulina velutipes [29].
Endogenous reference genes of different species have been reported. The endogenous reference gene for P. eryngii has not been reported. In the present study, s9ap was selected as the endogenous reference gene of P. eryngii by gene sequence alignment and blast analysis. Qualitative and quantitative PCR primers were designed for this gene, and the primers were verified and screened. The detection limit of SYBR quantitative PCR was 400 pg. The results show that s9ap gene was suitable for the detection of P. eryngii, and the specificity and detection limit both are good. The endogenous reference gene detection technology was an effective detection technology for wild mushroom adulteration.

DNA extraction and purification
The genomic DNA was extracted from fresh mushrooms by using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon, Shanghai, China). 100 mg of fresh mushroom fruiting bodies were first mixed with an equal amount of silica, ground into a powder with liquid nitrogen, and then extracted according to the kit instructions. The concentration and purity of all DNA samples were measured using Nano Drop2000 spectrophotometer (Thermo Scientific, Waltham, USA). The DNA integrity of the samples was verified by 1% agarose gel electrophoresis with ethidium bromide in 1× TAE buffer. DNA was amplified with universal fungi ITS primer, and the products were analyzed with 2% agarose gel electrophoresis with ethidium bromide in 1× TAE buffer. The DNA was stored at 4 °C for further use.

Oligonucleotide primers
All oligonucleotide primers used in this study were designed using ABI Prism Primer Express version 3.0 (Applied Biosystems, Foster City, USA) and synthesized by Shanghai Sangon Co. Ltd. (Shanghai, China). The designed primers were analyzed by BLAST alignment in NCBI to ensure the specificity. SYBR fluorescent dye kit was obtained from Tiangen Biochemical Technology Co. Ltd. (Tiangen, Beijing, China). ITS rDNA-F/R primers were used to evaluate DNA quality, and s9ap-F/R primer was used for the qualitative and quantitative PCR detection of s9ap gene. The detailed nucleotide sequences of the primers are listed in Table 1.

PCR conditions
The final volume of conventional PCR reaction was 25 µL as determined using ABI SimpliAmp thermal cycler (Applied Biosystems, USA). A single reaction system contained 2. The amplification products were analyzed by 2% agarose gel electrophoresis. The amplification procedure of the specific primer s9ap was the same as that of ITS except that the annealing temperature was 60 °C.

Selection of the candidate P. eryngii endogenous reference gene
The selection of appropriate endogenous reference gene can result in the accurate and practical detection of P. eryngii.
A good endogenous reference gene should be species-specific, with low and consistent interspecific copy numbers. In the nucleotide database of NCBI, the gene information of P. eryngii was searched. Through BLAST alignment and homology analysis, the s9ap gene for serine aminopeptidase (accession number: AB918644) with the lowest homogeneity with the sequences of the non-P. eryngii species was finally screened out as the alternative endogenous reference genes to be identified. The BLAST result and detailed information of s9ap are provided in Fig. S1. According to the gene sequence, species-specific primers were designed for qualitative and quantitative PCR.

Species specificity of the qualitative PCR assays
We extracted the DNA of 19 (Fig. 1), and ITS-F/R primers were used to detect the quality of the extracted DNA (Fig. 2).
The species specificity of s9ap gene in P. eryngii was qualitatively identified by conventional PCR amplification by using s9ap-F/R primers. The results showed that s9ap gene was successfully amplified in P. eryngii genomic DNA with obvious bands, while the other mushroom species had no amplification products, indicating that s9ap gene had high species specificity for P. eryngii (Fig. 3). than that of the negative control, indicating that the detection limit of SYBR Green I quantitative PCR was 80 pg/ µL (Fig. 4b). We established a standard curve of s9ap by using the quantitative PCR system. A linear relationship (R 2 = 0.98, slope = − 0.732) was determined between the DNA quantities and Ct values (Fig. 4c). These results indicate that the established s9ap SYBR Green I real-time PCR system was suitable for the accurate quantitative detection of P. eryngii.

Application of s9ap to examine different processed mushroom products
The established real-time PCR system was used to detect two kinds of mushroom products, namely, Fansaoguang (yexiangjun) and spicy P. eryngii. The DNA extracted from fresh P. eryngii was used as a positive control to ensure

Sensitivity of the qualitative and SYBR Green I realtime quantitative PCR assays
To detect the sensitivity of qualitative PCR, we gradient diluted the DNA concentrations from 15 ng/µL to 0.96 pg/ µL (five-fold serial dilutions) for amplification. The results showed that when the DNA concentrations were 15 and 3 ng/µL, clear specific bands were observed on the electrophoretic diagram, and their brightness gradually decreased. However, when the DNA concentration was reduced to 0.6 ng/µL, no amplification products were detected. Therefore, the detection limit of qualitative PCR was 3 ng/µL (Fig. 4a).
For the sensitivity of SYBR Green I real-time quantitative PCR, the genomic DNA concentrations were gradient diluted to 40, 4, 0.4, 0.08, and 0.016 ng/µL. When the DNA concentration was 80 pg/µL, specific amplification could still occur, and the CT value was significantly lower wall structure and are rich in polysaccharides, their DNA is difficult to extract, which greatly affects the efficiency of the whole experiment and limits the application and promotion of this method in actual sample detection.
In this study, we selected s9ap gene as the candidate endogenous reference gene of P. eryngii, and the specificity and sensitivity of s9ap gene were evaluated. The results indicated that a set of qualitative and quantitative PCR method was established for the detection of P. eryngii. However, electrophoresis results showed that the amplification efficiency of specific primers for the endogenous reference gene screened in this study was not ideal, the brightness of the electrophoretic bands was insufficient, this may be because the primers designed for the endogenous reference gene were not good enough, or there may be more ideal endogenous reference gene had not been screened. the practical use of s9ap. We amplified the DNA templates extracted from food samples with s9ap-F/R in conventional PCR (Fig. 5). Based on the results, no component of P. eryngii was observed in Fansaoguang (yexiangjun), but it could be detected in the spicy P. eryngii. The detection result was consistent with the food labels of the processed products.

Discussion
One challenge that most nucleic acid methods face is to combine the three key steps of molecular diagnostics, the three steps are sample preparation, amplification and detection, especially sample preparation [30]. In this study, mushrooms DNA were extracted by a kit method, the pretreatment steps were cumbersome. Because fungi have complex cell In conclusion, this method has the advantages of high sensitivity, high specificity, simple operation, low cost, and easy observation of results. It has a good development prospect for the rapid biological source detection of P. eryngii and its deep-processed products. It also provides the reference for the establishment of a set of qualitative and quantitative PCR method of other mushrooms based on endogenous reference gene.