Fluorescent quantitative PCR (Polymerase Chain Reaction) detection primer and probe for Chinese softshell turtle adenovirus as well as detection method and application thereof

Abstract

The invention relates to the technical field of adenovirus detection, and particularly discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) detection primer and probe for Chinese softshell turtle adenovirus, and nucleotide sequences of an upstream primer, a downstream primer and the probe are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the invention also discloses application of the primer and the probe in detecting the Chinese softshell turtle adenovirus and a kit comprising the primer and the probe. The invention also provides a detection method of the Chinese softshell turtle adenovirus. The detection method comprises the following steps: extracting DNA of a sample to be detected; the DNA is used as a template, and a primer and a probe are used for amplification; and judging whether the to-be-detected sample contains the Chinese softshell turtle adenovirus or not according to an amplification result. The qPCR primer and the probe have the beneficial effects that the qPCR primer and the probe have relatively high specificity on PsAdV, the Chinese softshell turtle adenovirus can be quantitatively detected, the sensitivity is high, and the detection method is simple, convenient, sensitive and high in specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of adenovirus detection, and in particular to a fluorescent quantitative PCR detection primer and probe for Chinese soft-shell turtle adenovirus, and a detection method and application thereof. Background Art

[0002] Chinese soft-shell turtle, Pelodiscus sinensis or Trionyx sinensis , belongs to the class Reptilia, order Testudinata, family Testudinidae, genus Testudinidae. It is widely distributed in China, Japan, Thailand and other countries. Due to its delicious taste and rich nutrition, the production of Chinese soft-shelled turtle has increased year by year, and it plays an important role in aquaculture in southern and central China. However, Chinese soft-shelled turtle farming is seriously affected by diseases. Because there are many types of pathogens that infect turtles, involving a variety of viruses and bacteria, the clinical symptoms are also relatively similar.

[0003] The Adenoviridae family is a non-enveloped icosahedral virion containing a linear dsDNA genome of 25-48 kb. Its members infect a variety of vertebrate hosts from fish to humans and are divided into six different genera: Adenine-thymine-rich adenovirus (Atadenovirus), Aviadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus and Testadenovirus (Benkő et al., 2022; Harrach et al., 2019). Among them, the strains of the genus Atadenovirus occur in sea turtles, the strains of the genus Aviadenovirus occur in fish, and the strains of the genus Siadenovirus infect birds, frogs and tortoises (Doszpoly et al., 2013; Doszpoly et al., 2019; Harrach et al., 2019). The severity of adenovirus infection ranges from subclinical to fatal (Benkő et al).

[0004] In 2023, the number of Chinese soft-shelled turtles ( Pelodiscus sinensis) has a new disease with low egg production, bleeding in the intestine and sternum epithelium, and undigested beige rubbery food residues in the intestine. The results showed that the cause of the disease was caused by a newly discovered adenovirus, tentatively named Chinese soft-shell turtle adenovirus (PsAdV-zg). Tian et al. analyzed the genome sequence of the virus and showed that the virus had a double-stranded DNA genome of 25548 bp, suggesting that it should be classified as a sialidase adenovirus (Siadenovirus) (Tian et al., 2025). Before this evidence was discovered, adenovirus infection had only been reported in three turtles and tortoises: Leopard tortoises (Wilkinson., 2004); Sulawesi tortoises (Rivera et al., 2009) and box turtles (Farkas et al., 2009). Among them, based on phylogenetic analysis of partial DNA polymerase gene sequences, the Sulawesi turtle adenovirus appears to be related to the genus Sialidadenovirus, which causes severe systemic disease with a very high mortality rate (Rivera et al., 2009).

[0005] From the perspective of preventing major aquaculture losses, it is necessary to establish a diagnostic method for the disease as soon as possible to effectively investigate the epidemiology of the disease and meet the requirements of timely prevention and control. Rapid on-site diagnosis of animal diseases is conducive to timely detection and control of animal diseases. However, it is difficult for front-line technicians to make an accurate diagnosis based on clinical symptoms, so how to quickly and accurately detect the pathogens of Chinese soft-shelled turtles is particularly important.

[0006] Real-time quantitative PCR (qPCR) mainly detects the amount of gene amplification by capturing fluorescent signals. It is a quantitative detection PCR technology with the characteristics of high detection sensitivity, strong specificity, and fast detection speed (Song et al., 2018). It is the most common technology for detecting and quantifying aquatic animal pathogens in the 2023 edition of the Manual of Diagnostic Tests for Aquatic Animals (World Organization for Animal Health, WOAH). Summary of the invention

[0007] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a fluorescent quantitative PCR detection primer and probe for Chinese soft-shelled turtle adenovirus and a detection method and application thereof.

[0008] The technical solution of the present invention is as follows: The first aspect of the present invention provides a fluorescent quantitative PCR detection primer for Chinese soft-shelled turtle adenovirus, comprising an upstream primer PsAdV-qPCR-F and a downstream primer PsAdV-qPCR-R; The nucleotide sequence of the upstream primer PsAdV-qPCR-F is shown in SEQ ID NO: 1; The nucleotide sequence of the downstream primer PsAdV-qPCR-R is shown in SEQ ID NO:2.

[0009] The second aspect of the present invention provides a Chinese soft-shell turtle adenovirus fluorescent quantitative PCR detection probe, including a probe PsAdV-qPCR-P, wherein the nucleotide sequence of the probe PsAdV-qPCR-P is shown in SEQ ID NO:3.

[0010] Optionally, the 5′ end of the probe PsAdV-qPCR-P is connected to a fluorescent reporter group FAM, and the 3′ end of the probe PsAdV-qPCR-P is connected to a fluorescent quencher group TAMRA.

[0011] The third aspect of the present invention provides the use of the primers and the probes in detecting Chinese soft-shelled turtle adenovirus.

[0012] The fourth aspect of the present invention provides the use of the primers and the probe in the preparation of a product for detecting Chinese soft-shelled turtle adenovirus.

[0013] The fifth aspect of the present invention provides the use of the primers and the probes in preparing a kit for detecting Chinese soft-shelled turtle adenovirus.

[0014] The sixth aspect of the present invention provides a fluorescent quantitative PCR kit for detecting Chinese soft-shelled turtle adenovirus, comprising the primers and the probes.

[0015] Optionally, the kit further comprises: 2×probe qPCR mix.

[0016] The seventh aspect of the present invention provides a method for detecting Chinese soft-shelled turtle adenovirus using the primers and probes, comprising the following steps: S1. Extract DNA from the sample to be tested; S2, using the DNA extracted in step S1 as a template, amplifying the primers, the probe and 2×probe qPCR mix under preset conditions; S3. According to the amplification result, determine whether the sample to be tested contains Chinese soft-shelled turtle adenovirus.

[0017] Optionally, the reaction system includes: 10 μL 2×probe qPCR mix, 600 nM of the primer, 200 nM of the probe and 2 μL of DNA template; The preset conditions include: denaturation at 95°C for 1 min; then at 95°C for 15 s; and 55°C for 60 s, for 40 cycles.

[0018] The present invention has at least one of the following beneficial effects: The present invention is the first to establish a PsAdVqPCR detection method for epidemiological investigation and disease prevention of Chinese soft-shelled turtle adenovirus infection, and to establish a real-time quantitative polymerase chain reaction (qPCR) detection method based on the specific sequence of the viral DNA polymerase gene. The results showed that the qPCR primers and probes of the present invention have high specificity for PsAdV, and are comparable to soft-shelled turtle iridovirus, Chinese soft-shelled turtle hemorrhagic syndrome virus ( Trionyx sinensis hemorrhagic syndrome virus), Citrobacter freundii ( Citrobacter freundii )、Aeromonas vervetii( Aeromonas veronii )、Aeromonas hydrophila( Aeromonas hydrophila )、Morganella morganii( Morganella morganii ) and Vibrio cholerae ( Vibrio cholera ) No cross-reaction; High detection sensitivity, effective amplification even at low target concentrations, PsAdV qPCR detection plasmid pUC57-PsAdV sensitivity of 1.0×10 1 The method of detecting Chinese soft-shell turtle gland disease using the above primers and probes is simple, sensitive and highly specific.

[0019] The present invention also performed qPCR detection on 107 clinical samples of Chinese soft-shelled turtle. The qPCR detection results were consistent with the previously published adenovirus consensus nested PCR detection results. The qPCR detection method has a high accuracy in detecting PsAdV in clinical specimens. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows the specificity test results of PsAdVqPCR detection using total DNA extracted from pUC57-PsAdV, PsAdV-zg and other pathogens. Among them, line 1: pUC57-PsAdV; line 2: PsAdV-zg genomic DNA; line 3: STIV; line 4: TSHSV; line 5: Cf; line 6: Av; line 7: Ah; line 8: Mm; line 9: Vc; line 10: negative control.

[0021] Figure 2 evaluates the sensitivity of the PsAdV qPCR assay for detecting pUC57-PsAdV DNA. Lines 1-7: The dilution range of pUC57-PsAdV DNA is 1.0×10 6-1.0 copies / μL; Line 8: negative control.

[0022] Figure 3 shows the standard curve of qPCR.

[0023] Figure 4 The repeatability of PsAdV qPCR detection of pUC57-PsAdV DNA. Among them, lines 1-3: pUC57-PsAdV plasmid dilution concentration is 1.0×10 6 Detection result curve of copies / μL; Lines 4-6: pUC57-PsAdV plasmid dilution concentration is 1.0×10 3 Detection result curve of copies / μL; Line 7: blank control. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Example 1 1.1 Pathogens of Chinese soft-shelled turtle and clinical specimens PsAdV-zg (Tian et al. 2025), Citrobacter freundii (Cf) LCJY-002 (Tian et al. 2013), Aeromonas vermiformis (Av), Aeromonas hydrophila (Ah), and Morganella morganii (Mm) were isolated, identified, and preserved by our laboratory. Chinese soft-shell turtle iridovirus (STIV) and Chinese soft-shell turtle hemorrhagic syndrome virus (TSHSV) were generously provided by Dr. Liu Hong, Shenzhen Customs, China, and Dr. Liu Li, Institute of Hydrobiology, Zhejiang Academy of Sciences, China, respectively. Vibrio cholerae (Vc) was provided by Nanchang Center for Disease Control and Prevention, Jiangxi Province.

[0026] A total of 107 clinical visceral tissue samples (liver, spleen, and kidney) of diseased Chinese soft-shelled turtles were collected from a Chinese soft-shelled turtle farm in Nanfeng County, Fuzhou City, Jiangxi Province, China from April to June 2024. The clinical features of these diseased turtles included skin rot, hepatomegaly, intestinal hemorrhage, intestinal obstruction, and mumps.

[0027] 1.2. DNA / RNA extraction and preparation of DNA standards Prepare 100 μL virus suspension: Take the liver, spleen and kidney tissues of the diseased turtle under sterile conditions, add PBS at a ratio of 1:5 and homogenize. Filter the tissue sample suspension filtrate through a 0.22 μm filter membrane and infect the turtle kidney cell line. After 5 days of infection, the cells show obvious pathological effects. After freezing and thawing, take 100 μL of the supernatant to prepare 100 μL virus suspension.

[0028] Prepare 200 μL of tissue sample suspension: take the liver, spleen, and kidney tissues of the diseased turtle, add PBS at a ratio of 1:1 to homogenize, and take 200 μL of the supernatant, which is the prepared 200 μL tissue sample suspension.

[0029] TaKaRa MiniBEST Viral RNA / DNA Extraction Kit (Beijing, China) was used to extract total DNA / RNA of all viruses from 100 μL of virus suspension according to the kit instructions. TaKaRa PrimeScript™ First-Strand cDNA Synthesis Kit (Beijing, China) was used to synthesize RNA viral cDNA according to the kit instructions. Bacterial genomic DNA was extracted using TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit (Beijing, China). Total DNA of clinical visceral tissues (liver, spleen, and kidney) of diseased Chinese soft-shelled turtles was extracted from 200 μL of tissue sample suspension using the Qiagen DNeasy Blood&tissueKit (Hilden, NRW, Germany).

[0030] The DNA polymerase gene fragment (3324 bp) of PsAdV-zg (GenBank accession number: PQ083072) was synthesized by Sangon Biotech, Shanghai, China, and cloned into the pUC57 vector, named pUC57-PsAdV plasmid standard. The pUC57-PsAdV standard plasmid DNA was extracted using the TIANGEN TIANprep Mini plasmid Kit (Beijing, China), and then the concentration was determined using the BioPhotometer Plus Nucleic Acid / Protein Analyzer (Eppendorf, Germany). The DNA copy number was calculated as follows: DNA copy number = (M× 6.02 × 10 23 × 10 -9 ) / (n × 660), the result is 1.0×10 9 Then, TE buffer was used to convert the concentration of 1.0×10 9 The pUC57-PsAdV standard plasmid DNA with 10 copies / μL was serially diluted 10 times. The extracted pUC57-PsAdV standard plasmid DNA and the serially diluted pUC57-PsAdV standard plasmid DNA were then stored at -80°C for later use.

[0031] 1.3. Design of qPCR primers and probes In order to design primers and probes targeting specific conserved DNA fragments in the PsAdV-zg genome, the present invention selected the DNA polymerase gene from the whole genome sequence of PsAdV-zg (GenBank accession number: PQ083072). Primer Premier 5.0 was used to design forward and reverse primers and probes for qPCR. All primers and probes were aligned by Blast to detect specificity and then synthesized by Sangon Biotech (Shanghai, China).

[0032] The primer and probe sequences designed by the present invention and the primer and probe sequences of the prior art "adenoviral consensus nested PCR" are shown in Table 1.

[0033] Table 1 Optimization of qPCR reaction conditions TaKaRa Probe qPCR Mix kit was used to perform TaqMan probe PsAdV qPCR amplification on Xi'an Tianlong Gentier 96E fluorescence quantification instrument. 7 100 copies / μL pUC57-PsAdV DNA was used as template, and a 20 μL reaction system was prepared according to the kit instructions. Amplification experiments were performed in the range of 50 ~ 62℃ with a gradient of 1℃ to determine the optimal annealing temperature; qPCR amplification was performed with different final concentrations of primers (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 μmol / L) and different final concentrations of probes (0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50 μmol / L), and the minimum critical cycle number (Ct) and relative fluorescence value (RFU) were compared to determine the optimal combination of primer and probe concentrations.

[0034] 1.5. qPCR specificity test The specificity of the established PsAdV qPCR detection method was evaluated by testing a variety of pathogens, including PsAdV-zg, STIV, TSHSV, Cf, Av, Ah, Mm, and Vc. 7 copies / μL) as the positive control, and ultrapure water as the negative control, and the results were repeated three times.

[0035] 1.6.qPCR sensitivity test To evaluate the sensitivity of qPCR detection of PsAdV, the concentration of 1.0× 10 6The detection limit of qPCR detection was determined by using the standard plasmid pUC57-PsAdV DNA standard diluted from 1.0 copies / μL to 1.0 copies / μL as the template, and the ultrapure water was used as the negative control template. The qPCR detection standard curve was generated according to the concentration gradient of the diluted plasmid, and the Ct value was calculated and expressed as mean ± standard deviation (SD).

[0036] 1.7.qPCR repeatability test At a concentration of 1.0×10 6 copies / μL and 1.0×10 3 The pUC57-PsAdV plasmid standard with 100 copies / μL was used as a template, and the PsAdV qPCR method established in this study was used for intra-group repeatability detection test, with 3 replicates for each standard concentration. Subsequently, 3 independent inter-group repeatability tests were performed under the same conditions every 7 days to evaluate the repeatability of the method.

[0037] 1.8. Practical application of clinical samples In order to evaluate the accuracy of the qPCR detection method established in the present invention for detecting clinical samples, 107 DNA samples were extracted from clinical visceral tissues (liver, spleen, and kidney) of diseased Chinese soft-shelled turtles with unknown PsAdV infection status. The extracted DNA was detected using the established detection method and the adenovirus universal nested PCR reported previously (Wellehan et al., 2004). The positive control, negative control, and blank control were pUC57-PsAdV plasmid DNA (1.0×10 7 copies / μL), DNA from healthy Chinese soft-shell turtle and DNA from ultrapure water.

[0038] 2. Results 2.1. Optimization results of PsAdV qPCR detection method The DNA polymerase gene sequence was selected as the target region for primer and probe design. Through optimization, the qPCR reaction was performed in a 20 μL reaction system, configured using the TaKaRa probe qPCR mix kit, which included: 10 μL 2×probe qPCR mix, 600 nM of each primer (PsAdV-qPCR-F / R), 200 nM TaqMan probe (PsAdV-qPCR-P) and 2 μL DNA template, and the reaction program was: denaturation at 95°C for 1 min, followed by 95°C for 15 s, 55°C for 60 s, and 40 cycles.

[0039] 2.2. Specificity test results When evaluating the specificity of the qPCR assay, no cross-reactions were observed with STIV, TSHSV, Cf, Av, Ah, Mm, Vc, and negative controls, and positive signals were only observed with PsAdV-zg genomic DNA and pUC57-PsAdV quality standards ( Figure 1 ). Therefore, the established PsAdV qPCR detection method has good specificity.

[0040] 2.3. Sensitivity test results Using 1.0×10 6 -1.0 copies / μL diluted recombinant plasmid was used to evaluate the sensitivity of the newly established qPCR detection method. Figure 2 As shown in Figure 2, the sensitivity of PsAdV qPCR for detecting plasmid pUC57-PsAdV was 1.0×10 1 copies / μL, indicating that the method of the present invention has high sensitivity and can effectively amplify even at low target concentrations.

[0041] Depend on Figure 2 It can be clearly seen that the amplification curve of qPCR moves backward regularly as the target concentration decreases. Figure 3 The qPCR standard curve showed that the plasmid concentration had a strong linear correlation with the Ct value (R 2 = 0.999), and the regression equation was Ct = −3.54 × lg (PsAdV DNA copy number) + 50.606, which confirmed the reliability of the established qPCR method to accurately quantify the target DNA through the conventional standard curve. In terms of reaction speed, qPCR takes about 60 min to obtain amplification results.

[0042] Repeatability analysis The PsAdV qPCR method established in the present invention was used, with a concentration of 1.0×10 3 The standard plasmid with 100 copies / μL was used as a template to verify the intra-group repeatability and inter-group repeatability. The results showed that the same concentration of template gave consistent results in the intra-group and inter-group tests of the method of the present invention, and the amplification curves changed very little (Figure 4). The results showed that the established PsAdV qPCR method had good reproducibility and high reliability.

[0043] 2.5. qPCR detection of PsAdV in clinical specimens The utility of the PsAdV qPCR assay was evaluated using 107 DNA samples extracted from clinical visceral tissue specimens of diseased Chinese soft-shelled turtles of unknown PsAdV infection status, and the results were then compared with those of the adenovirus universal nested PCR (Wellehan et al., 2004). Of the 107 samples, the adenovirus universal nested PCR assay gave positive results in 44 and negative results in 63. The results obtained from the assayed qPCR assay were consistent with those generated by the previously published adenovirus universal nested PCR, thus, the DSe and DSp of the established qPCR assay for clinical samples were both 100% compared with the adenovirus universal nested PCR.

[0044] Therefore, the positive rate of the newly established qPCR detection method for clinical specimens was 41.12% (44 / 107). The results showed that qPCR detection has a high accuracy in detecting clinical samples.

[0045] 3. Discussion In order to achieve laboratory quantitative analysis of PsAdV, the present invention aims to establish a reliable PsAdV qPCR detection method. Maximum likelihood analysis based on DNA polymerase amino acid sequences is one of the criteria recommended by the International Committee on Taxonomy of Viruses (ICTV) to distinguish adenovirus genera / species (Benkő et al., 2022). In order to improve the specificity of the detection method, DNA polymerase genes were selected to design primers and probes for the detection method. qPCR primers and probes were designed according to the design principles of qPCR primers and probes. The qPCR detection method of PsAdV was established by optimizing the annealing temperature, primer and probe concentrations. The logarithm of the concentration showed a good linear relationship with the Ct value, and the established PsAdV qPCR detection standard curve could accurately reflect the amplification of the target product.

[0046] The PsAdV qPCR detection method developed in the present invention has a high specificity for the detection of PsAdV, because only the standard plasmid pUC57-PsAdV and the DNA extracted from the Chinese soft-shelled turtle infected with PsAdV produced positive results on the amplification curve, which indicates that the developed PsAdV qPCR detection method has the potential to become a new differential diagnosis detection tool. The sensitivity test showed that the qPCR method can detect pUC57-PsAdV plasmid DNA with a detection value as low as 1.0×10 1 copies / μL. The qPCR standard curve showed a high correlation coefficient (R 2 =0.999), the regression equation is Ct=−3.54×lg(PsAdV DNA copy number)+50.606( Figure 3 ).

[0047] The qPCR of the present invention has higher sensitivity and stability in detecting trace target DNA. The qPCR technology is applied to the detection of trace pathogens in turtles, and is recommended for quantitative analysis in laboratory research. qPCR is more suitable for quantitative analysis of samples with low target concentrations.

[0048] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A fluorescent quantitative PCR detection primer for Chinese soft-shelled turtle adenovirus, characterized in that: Includes upstream primer PsAdV- qPCR -F and downstream primer PsAdV- qPCR -R; The nucleotide sequence of the upstream primer PsAdV-qPCR-F is shown in SEQ ID NO: 1; The nucleotide sequence of the downstream primer PsAdV-qPCR-R is shown in SEQ ID NO:

2.

2. A fluorescent quantitative PCR detection probe for Chinese soft-shelled turtle adenovirus, characterized in that: It includes a probe PsAdV-qPCR-P, the nucleotide sequence of which is shown in SEQ ID NO:

3.

3. The probe according to claim 2, characterized in that: The 5′ end of the probe PsAdV-qPCR-P is connected to a fluorescent reporter group FAM, and the 3′ end of the probe PsAdV-qPCR-P is connected to a fluorescent quencher group TAMRA.

4. Use of the primer according to claim 1 or the probe according to claim 2 or 3 in detecting Chinese soft-shelled turtle adenovirus.

5. Use of the primer according to claim 1 or the probe according to claim 2 or 3 in preparing a product for detecting Chinese soft-shelled turtle adenovirus.

6. Use of the primer according to claim 1 or the probe according to claim 2 or 3 in preparing a kit for detecting Chinese soft-shelled turtle adenovirus.

7. A fluorescent quantitative PCR kit for detecting Chinese soft-shell turtle adenovirus, characterized in that: It comprises the primer according to claim 1 and the probe according to claim 2 or 3.

8. The kit according to claim 7, characterized in that The kit also includes: 2×probe qPCRmix.

9. A method for detecting Chinese soft-shell turtle adenovirus using the primers according to claim 1 and the probes according to claim 2 or 3, characterized in that: The following steps are involved: S1. Extract DNA from the sample to be tested; S2, using the DNA extracted in step S1 as a template, amplifying the primers, the probe and 2×probe qPCR mix under preset conditions; S3. According to the amplification result, determine whether the sample to be tested contains Chinese soft-shelled turtle adenovirus.

10. The method according to claim 9, characterized in that The reaction system included: 10 μL 2×probe qPCR mix, 600 nM of the primer, 200 nM of the probe and 2 μL of DNA template; The preset conditions include: denaturation at 95°C for 1 min; then at 95°C for 15 s; and 55°C for 60 s, for 40 cycles.

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