Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

Enterocytospora artemiae (EAM) mainly parasitizes the hepatopancreas of Palaemonetes sinensis. Serious infection leads to hepatopancreatic lesions, which greatly reduce the vitality of P. sinensis. Currently, EAM is detected via conventional PCR methods. However, conventional PCR has low sensitivity and cannot be used for accurate quantitative detection of EAM or its parasitic activity in host tissues. In this study, we designed a pair of specific primers based on the sequence of the ribosomal protein S9 gene (RPS9; GenBank accession number: MZ420734) to establish and optimize a SYBR Green I real-time fluorescent quantitative PCR detection method for EAM. Only EAM appeared as a bright and single target band, whereas other microorganisms did not, indicating that the primer for RPS9 had high specificity. This method displayed optimum amplification effects at an annealing temperature of 55°C, and the melting curve of the product produced a single peak. The established method showed a good linear relationship from 2.2 × 108 to 2.2 × 101 copies/μL. The relationship between the number of cycle thresholds (Ct) and the logarithm of the initial template amount (x) conformed to Ct = −3.281 log x + 36.543 (R2 = 0.998). Amplification efficiency was 101.737%, and the lower limit of detection sensitivity was 2.2 × 101 copies/μL. Good intra- and inter-group repeatability was observed within the linear range. The sensitivity of this method was more than 200 times higher than that of nested PCR. Thus, detection data obtained using this method may be useful as a technical reference for rapid and accurate identification of EAM infection and for the prevention and control of EAM during P. sinensis breeding.


INTRODUCTION
Microsporidia are a group of common fungal intracellular parasites consisting of over 200 genera and 1600 species . This group has a wide range of hosts, including almost all lower invertebrates, as well as higher vertebrates. Microsporidia are opportunistic pathogens, which easily infect patients receiving immunosuppressive therapy for congenital immune disorders (Pol et al., 1993;Didier and Weiss, 2011). Currently, 17 microsporidia species belonging to nine genera are known for their ability to infect humans (Pan et al., 2018). In addition to causing harm to human health, many microsporidia cause economic damage to important animal and plant industries (Kent and Speare, 2005;Higes et al., 2013). For example, the pepper disease of silkworms, which plagued the silkworm industry for more than two centuries, is caused by Nosema bombycis, a microsporidian (Nageswara Rao et al., 2004). Decapoda, an order of crustaceans that hosts microsporidia, has been studied for more than 100 years. As early as the end of the nineteenth century, French researchers reported that Thelohania infects Palaemon rectirostris, P. serratus, Crangon vulgaris, Procambarus clarkii, and Astacus fluviatilis as well as other shrimp (Stentiford and Dunn, 2014). Reportedly, the infection rate as well as the transmission range of Enterocytozoon hepatopenaei (EHP) in Litopenaeus vannamei has been increasing annually, making it one of three major pathogens endangering healthy L. vannamei cultures (Biju et al., 2016;Chaijarasphong et al., 2020).
Enterocytospora artemiae (EAM) was first discovered in Artemia in 2013. It was considered as an obligate parasite of Artemia that was confined to Europe and America (Rode et al., 2013). However, in 2020, it was found that this microsporidian had spread to Asia and was infecting the economically important Chinese grass shrimp, Palaemonetes sinensis (Jiang et al., 2020). EAM mainly parasitizes the hepatopancreas of P. sinensis (Jiang et al., 2020). Serious EAM infections lead to hepatopancreatic lesions and greatly reduce the vitality of P. sinensis. Although the mortality rate is not high, absorption of host nutrition by these microsporidia leads to increased breeding costs. Microsporidia are minute and do not cause obvious symptoms after infecting a host. Therefore, it is necessary to establish a rapid and accurate method to detect these parasites. Currently, only conventional PCR methods are used to detect EAM (Rode et al., 2013;Jiang et al., 2020). However, conventional PCR has low sensitivity and cannot be used for accurate, quantitative detection of EAM or its parasitic activity in host tissues. When the number of microsporidia infecting a host is low, observation via traditional staining followed by microscopy or electron microscopy becomes ineffective, preventing various species of microsporidia from being accurately determined. In contrast, real-time fluorescence quantitative PCR (qPCR), which is associated with advantages such as high specificity as well as high sensitivity, consumes less time and allows the copy number of the initial DNA template in the sample to be quantitated. The qPCR detection process is completed in a relatively closed and independent system, which prevents cross contamination of the sample and environment (Kotková et al., 2018;Li et al., 2019). In this study, we designed a nested PCR method, leading to the establishment of a qPCR protocol that detects EAM using SYBR Green I fluorescent dye. We compared the sensitivity and detection efficacy of conventional PCR, nested PCR, and qPCR to ensure rapid and accurate identification of EAM infections to control and prevent infection during P. sinensis breeding.

MATERIALS AND METHODS
Sample Collection of Palaemonetes sinensis P. sinensis was collected from farms 1, 2, and 3 in Panjin, Liaoning province. These shrimp were transported to Shenyang Agricultural University for temporary cultivation. During the temporary cultivation period, 30 shrimp were randomly selected from each farm to detect the prevalence via different methods. During sampling, the live shrimp were anesthetized on ice for 5 min, and the hepatopancreases were removed quickly and placed in a centrifuge tube containing 100% alcohol for further analysis.

DNA Extraction
DNA was extracted from the tissues of P. sinensis in strict accordance with the instructions of the TIANamp Marine Animals DNA Kit (Tiangen, Beijing). The extracted DNA was diluted to 50 ng/µL after determination of DNA concentration with an ultra-microspectrophotometer (K5500, Beijing Kaiao Technology Development Co., Ltd.), and then was stored at −20 • C.

Conventional PCR Amplification and Nested PCR Primer Design
Previously reported universal 18S rDNA primers, V1F and 1492R, were used for PCR amplification (Rode et al., 2013). The V1F and 1492R sequences are listed in Table 1. The amplification procedure was as follows: denaturation at 94 • C for 3 min; 35 cycles (denaturation at 94 • C for 45 s, annealing at 45 • C for 30 s, and extension at 72 • C for 90 s); and a final extension at 72 • C for 5 min. The PCR reaction system contained a total of 15 µL, with 13 µL of 2 × Taq Plus Master Mix, 0.5 µL of Primer-F, 0.5 µL of Primer-R, and 1 µL of DNA template.
Primers, V1F, and 1492R were used for the first round of nested PCR amplification. The second-round primers, YW-F and YW-R, are shown in Table 1. The target segment was 322 bp. The

Preparation of Plasmid Standard
Conventional PCR amplification: Using the EAM-positive samples as the template, the ribosomal protein S9 (RPS9) primers were used for amplification. The primers specific for RPS9 (GenBank accession number: MZ420734), RPS9-F, and RPS9-R, were designed using Primer 5.0 (Supplementary File 1). The RPS9-F and RPS9-R primer sequences are shown in Table 1.
The PCR procedure was as follows: pre-denaturation at 94 • C for 3 min; 35 cycles (denaturation at 94 • C for 30 s, annealing at 55 • C for 30 s, and extension at 72 • C for 30 s); and a final extension at 72 • C for 5 min. The PCR reaction system was the same as that described for nested PCR.
Purification and recovery of DNA fragments: The PCR product was dispensed into the well of a 1.5% agarose gel, which was then transferred to an electrophoresis apparatus and run at 100 V for 38 min. After electrophoresis, a strong, positive single strip of 206 bp was rapidly excised from the gel, and the gel containing the target strip was placed in a centrifuge tube, following which DNA was recovered using a FastPure Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China).
Vector recombination: A reaction solution (5 µL total volume) composed of pMD-19-T vector (1 µL), extracted DNA samples (1 µL), and ddH 2 O (3 µL) was prepared. Next, 5 µL of solution I was added to the prepared reaction solution and left to stand overnight at 4 • C.
Strain screening: A mixture of vector solution and Escherichia coli DH5α competent cells was added to a 1.5 mL centrifuge tube, placed on ice for 30 min, heated at 42 • C for 45 s, and placed on ice for 1 min. Subsequently, 890 µL of LB medium was added, and the mixture was shaken at 37 • C for 60 min. Selection plates were made by adding 35 µL of X-gal and 5 µL of IPTG to LB agar medium containing ampicillin (AMP). Next, 100 µL of bacterial solution was spread on the selection plate, and the plate was placed in an incubator at 37 • C overnight after sealing with parafilm. A single white colony was picked from the plate using a pipette tip and dissolved in 100 µL of ddH 2 O. One microliter of the plasmid DNA sample was verified via sequencing (Sangon, Shanghai, China). The remaining liquid was poured into a centrifuge tube containing 10 mL of AMP-LB liquid and placed in an orbital shaker for approximately 4 h until the liquid was turbid. Plasmids were extracted using a FastPure Plasmid Mini Kit (Vazyme, Nanjing, China).

Optimization of the Quantitative PCR Amplification Conditions
The initial concentration of the plasmid was 7.3 ng/µL. According to the formula copies/µL = initial concentration (ng/µL) × 6.02 × 10 14 /[(base number of vector fragment + base number of target gene) × 660], the copy number of the recombinant plasmid was 2.2 × 10 9 copies/µL. The recombinant plasmid was diluted using a 10-fold serial dilution with DEPCtreated water, and a 2.2 × 10 8 -2.2 × 10 1 copies/µL plasmid standard was used as a template for qPCR amplification. The recombinant plasmid gradient was used as the standard, and the total reaction system volume was 20 µL. The PCR reaction system contained 10.0 µL of 2 × ChamQ Universal SYBR qPCR Master Mix, 0.4 µL of RPS9-F, 0.4 µL of RPS9-R, 8.2 µL of ddH 2 O, and 1 µL of recombinant plasmid. The blank group was treated with ddH 2 O instead of DNA samples. The amplification reaction was performed in the Applied Biosystems QuantStudio 3 (Thermo Fisher Scientific, Waltham, MA, United States). After pre-denaturation at 95 • C for 30 s, 40 cycles of amplification at 95 • C for 10 s, X • C for 30 s (X was the annealing temperature gradient and the set range was 50-65 • C) and 72 • C for 30 s were carried out to select the best annealing temperature. The data analysis was performed using QuantStudio Design and Analysis Software v1.4 (Applied Biosystems). The optimum annealing temperature was 55 • C, and the reaction procedure was as follows: pre-denaturation at 95 • C for 30 s and 40 cycles (95 • C for 10 s; 55 • C for 30 s; 72 • C for 30 s).

Plotting of Quantitative PCR Standard Curve for Enterocytospora artemiae
The recombinant plasmid was diluted via a 10-fold serial dilution, which yielded a dilution series of 8 concentrations ranging from 2.2 × 10 8 to 2.2 × 10 1 copies/µL. The standard curve of the relationship between the logarithm of the initial quantity of the standard template (x) and the cycle threshold number (Ct) of the amplification products was established. The quality of the standard curve was determined by analyzing the correlation coefficient and amplification efficiency.

Sensitivity Comparison, Specificity Analysis, and Stability Evaluation of Quantitative PCR Primer RPS9
The sensitivity of qPCR and conventional PCR (RPS9), indicated by recombinant plasmids with a concentration of 2.2 × 10 8 -2.2 × 10 1 copies/µL, and detection limits for qPCR and conventional PCR were analyzed by determining whether showing an S-shaped amplification curves and whether the target bands appeared separately. Furthermore, qPCR, nested PCR (V1F/1492R-YW-F/YW-R), and conventional PCR (V1F/1492R) were used to detect the 90 shrimp hepatopancreas samples simultaneously.
DNA from the hepatopancreases of healthy P. sinensis was used as the negative control, and microsporidia (EHP, Hepatospora eriocheir and Microsporidia sp.), viruses (white spot syndrome), bacteria (Pseudomonas putida and Vibrio parahaemolyticus), and fungi (Metschnikowia bicuspidata) were detected simultaneously. These pathogens were obtained from our laboratory. Specificity was analyzed using conventional PCR (RPS9) and qPCR.
To determine the stability of qPCR, serially diluted positive plasmid DNA (2.2 × 10 8 -2.2 × 10 1 copies/µL) was used for qPCR templates, in which three replicates were set for each template concentration. Stability assessment consisted of two parts: intra-group repetition (in the same experiment) and intergroup repetition (in the same group). For intra-group repetition, the mean value, standard deviation, and coefficient of variation were calculated using the Ct value of three replicates (Liu et al., 2016). For inter-group repetition, the experiment was repeated thrice. The mean value, standard deviation, and coefficient of variation were calculated using the Ct value of three experiments (Ding et al., 2017).
Tissue Tropisms and Detection of Potential Enterocytospora artemiae Carriers by Quantitative PCR Tissue tropisms: 10 shrimp were randomly selected from the temporary container for tissue tropism detection. The selected tissues included the hepatopancreas, gut, gill, muscle, and stomach.
Potential carriers: Biological and water samples were collected from the rice field of infected P. sinensis. The biological samples included fish, two Chanodichthys erythropterus and five Pseudorasbora parva; shrimp, five Macrobrachium nipponense and four Neocaridina denticulate; five cladocerans; and five copepods. The hepatopancreas tissues of fish and shrimp and the whole cladocerans and copepods were used for DNA extraction. The water samples from the rice field were filtered using a 0.22 µM filter membrane. The filter membrane was then washed with distilled water, the supernatant was discarded after centrifugation (8000 rpm, 10 min), and the precipitate was used to extract DNA. The extracted DNA was diluted to 50 ng/µL, and then was detected by qPCR and nested PCR. PCR amplification products of nested PCR

Specificity Analysis
The specificity of qPCR primers was analyzed by detecting other microsporidia, fungi, bacteria, and viruses. Only the hepatopancreatic DNA of EAM-infected shrimp appeared as a bright, single target band (Figure 1A), indicating that the primer RPS9 had high specificity. In addition, the qPCR amplification curve showed that only samples with positive EAM showed amplification curves, whereas no fluorescence signal was detected for non-target genes (Figure 1B), further indicating that the primer showed excellent specificity.

Construction of Quantitative PCR Standard Curve
The established qPCR system for EAM was used to amplify eight standard templates (2.2 × 10 8 -2.2 × 10 1 copies/µL) with 10-fold gradient dilution, and the standard curve of the relationship between the threshold Ct value and the logarithm of the initial quantity of standard templates (x) was established. The qPCR results showed a strong fluorescence signal from 2.2 × 10 8 to 2.2 × 10 1 copies/µL (Figure 2A). The relationship was Ct = − 3.281 log x + 36.543 ( Figure 2B). The correlation coefficient R 2 was 0.998, and the amplification efficiency was 101.737%.
Melting curve analysis ( Figure 2C) showed that the curves of three parallel amplification products in each gradient group merged to produce a single melting peak at 82.4 • C, with overlapping positions. This unique melting peak indicated that there was no non-specific amplification or primer dimer formation during the amplification process.

Stability Assessment
Six concentrations of plasmid (2.2 × 10 8 -2.2 × 10 1 copies/µL) were selected, and a repeatability analysis was conducted using three parallel samples of the same gradient within the group. The coefficient of variation of Ct was much less than 1% (Table 2), indicating that intra-group repeatability was very good. All coefficients of variation between groups were less than 2% (Table 2), indicating that the method showed good repeatability, thereby ensuring the stability and reliability of the test results.

Detection of Samples by Nested PCR and Quantitative PCR for Enterocytospora artemiae
Nested PCR showed that, of the 90 P. sinensis hepatopancreas samples, 56 were positive, indicating a detection rate of 62.22% (56/90). The ratio of PCR samples negative in the first round to PCR samples positive in the second round was 37/56 (66.07%), and the ratio of positives in both rounds was 19/56 (33.93%). The qPCR results showed that 73 shrimp samples were positive, with a detection rate of 81.11% (73/90). Ct was substituted in the established formula Ct = − 3.281 log x + 36.543. The E. artemiae content ranged from 4.4 × 10 1 to 6.3 × 10 7 copies/µL. EAM could be detected only when the level of 1.4 × 10 5 copies/µL was exceeded in the first round of PCR. When the copy number of EAM small subunit (SSU) rRNA was lower than 4.6 × 10 3 copies/µL, the results for nested PCR were negative, whereas the limit of qPCR for EAM was 2.2 × 10 1 copies/µL. This indicates that the sensitivity of qPCR for EAM was over 200 times higher than that of nested PCR for detecting P. sinensis in hepatopancreas samples.

Tissue Tropisms and Potential Carrier Detection by Quantitative PCR for Enterocytospora artemiae
The EAM load in the five tissues differed ( Table 3). It was highest in the hepatopancreas, followed by the gut, gill, stomach, and muscle. However, there was no difference in the positive rate of EHP among the five tissues, all of which were 80%.
The water samples collected from the rice field of infected P. sinensis were all positive by qPCR detection. Some samples of fish and shrimp in the collected aquatic biological samples were positive by PCR detection, and the positive rate of cladocerans and copepods reached 80% (Table 4). However, C. erythropterus and M. nipponense was not detected by nested PCR (Table 4). Nested PCR positive samples were confirmed as EAM after sequencing and blast analysis, indicating that qPCR detection method had high sensitivity.

DISCUSSION
Rapid and accurate identification of pathogens is the basis of aquatic animal disease control. Microscopic examination is a rapid method. However, microsporidia can be observed under high magnification only when infection is severe. In addition, it is difficult to accurately identify microsporidia only by appearance due to inconsistencies during different developmental stages. Therefore, it is impossible to obtain accurate information regarding E. artemiae infection via microscopic examination. Based on this, highly sensitive molecular methods, such as PCR (Tang et al., 2015), nested PCR (Jaroenlak et al., 2016), qPCR (Liu et al., 2018;Piamsomboon et al., 2019), loopmediated isothermal amplification (Suebsing et al., 2013;Cai et al., 2018), and recombinase polymerase amplification (Ma et al., 2021), are used instead of microscopic methods. Compared with conventional PCR, qPCR has the advantages of high sensitivity, good repeatability and quantification. Furthermore, qPCR reduces non-specific amplification and shortens detection time. Two common qPCR methods are the TaqMan probe and the SYBR Green I fluorescent dye method. Although the TaqMan probe method shows higher specificity, its cost of detection is also high, limiting its practical use. The SYBR Green I fluorescence method has the advantages of low cost and easy implementation and is more suitable for rapid detection. Notably, qPCR is often used in the detection of microsporidia and is considered to be more sensitive than conventional PCR (Naung et al., 2020). Wang et al. (2020) used qPCR to detect the polar tube protein 2 gene when quantitatively analyzing EHP in infected shrimp and reported that sensitivity was increased by at least two orders of magnitude compared with that of conventional PCR. Phelps and Goodwin (2007) used the same technology to detect Notemigonus crysoleucas and reported that the minimum gene copy number that could be detected in each reaction was as low  as 10. Because the SSU rRNA gene of microsporidium is not a single-copy gene, ultrasonic treatment of spores can be used to extract DNA, which improves detection sensitivity, enabling 0.14 spores to be detected at the lowest level in each reaction (Phelps and Goodwin, 2007). In this study, the sensitivity of qPCR for detecting EAM was higher than that of conventional PCR and nested PCR. With real samples, nested PCR did not detect target DNA fragments below 4.6 × 10 3 copies/µL, and the sensitivity was only approximately 1/200 of that of qPCR for EAM. In contrast, in a batch of real samples, the detection rate of qPCR for EAM (81.11%) was significantly higher than that of nested PCR (62.22%). Meanwhile, the qPCR method was used to detect EAM tissue tropism and the carriers in the culture environment. The tissue tropism detection found that the content in the hepatopancreas was the highest, indicating that it was the main location of EAM, which was consistent with previous research (Rode et al., 2013;Jiang et al., 2020). At the same time, EAM was also detected in the gut, gill, stomach and muscle, and the same results have been obtained with EHP (Santhoshkumar et al., 2017;Cheng et al., 2018). Although EAM was detectable in these tissues in this study, whether EAM can infect the cells of these tissues needs to be further studied. Furthermore, the same infection rates were obtained in different tissues, which may be related to the high content of EAM in P. sinensis. Certainly, hepatopancreatic tissue was preferred because the load was the highest (Table 4). However, when hepatopancreas tissue is used for the determination of other indexes in research, tissues such as the gut can be considered as an alternative for detecting infection. Environmental detection found EAM in some biological samples, except for Neocaridina denticulata, and the detection rate in water was 100%. Sequencing showed that the positive sample of nested PCR was EAM, but the detection rate was significantly lower than qPCR, indicating that qPCR method has high sensitivity and can find carriers in the environment. This can provide guidance for transmission route research and prevent EAM infection.
Researchers have attempted to use qPCR to detect different microsporidia genes, such as those encoding the SSU rRNA, the polar tube protein, tubulin, and the spore wall protein (Jaroenlak et al., 2016;Han et al., 2018;Piamsomboon et al., 2019;Wang et al., 2020). Of these, the SSU rRNA gene carries the most commonly used detection target sequence. However, this gives rise to false positive results and shows high similarity with SSU rRNA genes of different species, leading to false detections (Lucchi et al., 2012;Tangprasittipap et al., 2013;Hitakarun et al., 2014). Sequence alignment showed that the EAM SSU rDNA gene (GenBank No. MT 645708) had 94% shared homology with the SSU rRNA gene of Globulispora mitoporans (GenBank No. KT762153), which shared more than 85% identity with five other species (Jiang et al., 2020). Therefore, we selected a specific target to modify the SYBR Green I qPCR detection method to detect microsporidia in P. sinensis. In this study, we established a qPCR method for the detection of EAM using RPS9 and established a minimum copy number of 22 copies/µL, indicating a highly sensitive diagnostic standard. RPS9 is widely distributed in yeast, bacteria, parasites, mammals, and humans. Studies have shown that RPS9 is a highly conserved gene, which plays a role in DNA repair, self-translation regulation, development regulation, malignant transformation of normal cells as well as in other undetected functions and is often used for pathogen detection (Lindström and Zhang, 2008;Lindström and Nistér, 2010;Araújo et al., 2012). In addition, whereas three species of microsporidia were detected using this study, no specific bands or interference reactions pertaining to other shrimp pathogens were found (Figure 2), indicating that the qPCR-RPS9 method showed high specificity.
Establishment of a qPCR method specifically for microsporidium not only enabled microsporidia to be detected sensitively and efficiently, but also allowed quantification. Analyzing microsporidium load is extremely important when conducting research on the prevention and control of microsporidia. Liu et al. (2016) reported that EHP load was negatively correlated with the growth of L. vannamei. When the relative copy number of EHP SSU rDNA in the hepatopancreas exceeded 10 3 copies/ng hepatopancreatic DNA, the growth of the shrimp was significantly slower, indicating that the effect of EHP on shrimp growth had reached a high-risk level (Liu et al., 2016). Thus, detection of microsporidium load enables better breeding strategies to be formulated. A serious microsporidium infection in P. sinensis causes body color turbidity and hepatopancreatic lesions, seriously reducing aquaculture yields (Jiang et al., 2020). Therefore, establishment of a rapid and quantitative detection method may help determine the severity of infection. In addition, this method may be useful for clarifying the relationship between EAM load and growth, leading to productive P. sinensis cultivation.

CONCLUSION
Serious EAM infection leads to hepatopancreatic lesions, which greatly reduce the vitality of P. sinensis. Conventional PCR has low sensitivity and cannot be used for accurate quantitative detection of EAM. In this study, a SYBR Green I real-time fluorescent quantitative PCR detection method based on the sequence of the ribosomal protein S9 gene was established and optimized. This method had high specificity and stability and was more than 200 times more sensitive than nested PCR. Thus, detection data obtained using this method may be useful as a technical reference for rapid and accurate identification of EAM infection and for the prevention and control of EAM during P. sinensis breeding.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Experiments Ethics Committee of Shenyang Agricultural University.

AUTHOR CONTRIBUTIONS
HJ, JB, and QC were involved in designing of the research and wrote the manuscript. HJ, JB, JL, YC, and SH performed the majority of the experiment, data processing, analysis, and interpretation. CF and XL assisted in sample collection and wrote the manuscript. All authors contributed to the article and approved the submitted version.