Pelteobagrus fulvidraco calicivirus and streptococcus iniae dual fluorescent quantitative PCR (polymerase chain reaction) method

Abstract

The invention discloses a pelteobagrus fulvidraco calicivirus and streptococcus iniae dual fluorescent quantitative PCR method, and belongs to the technical field of biological diagnosis. The invention provides a pelteobagrus fulvidraco calicivirus and streptococcus iniae dual detection primer and a fluorescent probe. The pelteobagrus fulvidraco calicivirus and streptococcus iniae dual detection primer comprises primers as shown in SEQ ID NO.1-2 and SEQ ID NO.4-5, and the fluorescent probe comprises fluorescent probes as shown in SEQ ID NO.3 and SEQ ID NO.6. According to the present invention, the rapid and accurate diagnosis of the two pathogens is achieved by amplifying and detecting the two nucleic acids such as the pelteobagrus fulvidraco calicivirus and the streptococcus iniae by using the principle of the fluorescent quantitative PCR technology, and the lowest detection limits of the YcCV and the S. iniae are respectively 1 * 100 copies / [mu] L and 1 * 100 copies / [mu] L by using the detection method provided by the present invention. Important technical support is provided for prevention and control of diseases of the pelteobagrus fulvidraco.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological diagnosis, and particularly relates to a duplex fluorescent quantitative PCR method for yellow catfish calicivirus and streptococcus iniae. BACKGROUND

[0002] In the detection of streptococcus iniae, the PCR diagnosis method of specific primers has been successfully applied. This method has very high sensitivity, and the detection limit can reach the level of a single bacterium, and can effectively detect pathogenic bacteria in tissue homogenate. In addition, the phylogenetic tree constructed based on ribosomal ribonucleic acid (rRNA) gene has become an important tool for studying the evolutionary relationship and affinity of bacteria. The rRNA of bacteria is composed of 5S, 16S and 23S, which correspond to rrs, rrl and rrf genes respectively. Among them, the structure of 16S rRNA and 23S rRNA both contain conserved regions and variable regions, and the sequence of the conserved region is less different between different bacteria, while the sequence of the variable region is significantly different with the closeness of the genetic relationship of bacteria, which provides an important basis for bacterial identification.

[0003] As the core method of molecular diagnosis, PCR technology plays an important role in pathogen detection by specific amplification of target gene fragments. For the detection of streptococcus iniae, researchers usually choose 16S rRNA gene, gyrB gene and other conserved regions to design primers. The application of nested PCR and fluorescent quantitative PCR (qPCR) and other technologies further improves the sensitivity and specificity of detection. Especially the qPCR technology, with its high sensitivity, accuracy and real-time quantification, is widely used in pathogenic bacteria detection field. qPCR can not only detect the presence of target DNA, but also can be quantitatively analyzed, which provides an important basis for disease assessment and efficacy monitoring. In addition, qPCR has the advantages of high automation degree and simple operation, which is suitable for large-scale sample detection.

[0004] In the detection of viruses, calicivirus, as a class of single-stranded RNA viruses with envelope, its rapid evolution characteristic brings great challenge to traditional detection methods. The early clinical symptoms of calicivirus infection (such as body surface hemorrhage and reduced feeding) lack pathogen specificity, which is easy to be confused with bacterial sepsis, leading to misdiagnosis and delay of prevention and control. The genome of calicivirus shows high variability, and the traditional antibody detection method often shows sensitivity decay due to epitope drift, while the conventional RT-PCR is difficult to achieve broad-spectrum detection due to the difficulty of primer design targeting the conserved region. SUMMARY

[0005] The purpose of the present application is to provide a duplex fluorescent quantitative PCR method for yellow catfish calicivirus and streptococcus iniae, in order to solve the problems existing in the prior art.

[0006] To achieve the above object, the present application provides the following scheme: One of the technical solutions of the present application is a double detection primer and fluorescent probe for Pelteobagrus cup-shaped virus and Streptococcus iniae, wherein the detection primer comprises primers shown as SEQ ID NO. 1-2 and SEQ ID NO. 4-5, and the fluorescent probe comprises fluorescent probes shown as SEQ ID NO. 3 and SEQ ID NO. 6.

[0007] The second technical solution of the present application is a kit for double detection of Pelteobagrus cup-shaped virus and Streptococcus iniae, comprising the double detection primer and fluorescent probe for Pelteobagrus cup-shaped virus and Streptococcus iniae.

[0008] The third technical solution of the present application is a double detection method of Pelteobagrus cup-shaped virus and Streptococcus iniae for non-disease diagnosis purposes, which uses DNA of a sample to be tested as a template, and uses the double detection primer and fluorescent probe for Pelteobagrus cup-shaped virus and Streptococcus iniae to perform real-time fluorescent quantitative reaction, and determines whether the sample contains Pelteobagrus cup-shaped virus and Streptococcus iniae based on the Ct value of the template DNA.

[0009] The fourth technical solution of the present application is application of the double detection primer and fluorescent probe for Pelteobagrus cup-shaped virus and Streptococcus iniae in preparation of a product for detecting Pelteobagrus cup-shaped virus and Streptococcus iniae.

[0010] Based on the above technical solutions, the present application has the following technical effects: The present application uses the principle of fluorescent quantitative PCR technology, and realizes rapid and accurate diagnosis of the two pathogens by amplifying and detecting the two nucleic acids of Pelteobagrus cup-shaped virus and Streptococcus iniae. The minimum detection limit of the detection method provided by the present application for YcCV and S. iniae is 1x10 0 copies / μL and 1x10 0 copies / μL, respectively, which provides important technical support for prevention and control of Pelteobagrus diseases. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Figure 2 is the amplification results of YcCV and S. iniae genes. Among them, a: the amplification results of YcCV; b: the amplification results of S. iniae.

[0012] Figure 2 Figure 4 is the primer concentration test results. Among them, green: primer concentration test results of YcCV; blue: primer concentration test results of S. iniae.

[0013] Figure 3The results of the probe concentration test. Among them, green: the results of the probe concentration test of YcCV; blue: the results of the probe concentration test of S. iniae.

[0014] Figure 4 The results of the specificity experiment. Among them, green: YcCV TaqMan double real-time fluorescent quantitative PCR amplification curve; blue: S. iniae TaqMan double real-time fluorescent quantitative PCR amplification curve.

[0015] Figure 5 The results of the interference experiment. Among them, green: YcCV TaqMan double real-time fluorescent quantitative PCR amplification curve; blue: S. iniae TaqMan double real-time fluorescent quantitative PCR amplification curve.

[0016] Figure 6 The results of the YcCV sensitivity test. Among them, a: 1×10 0 ~1×10 7 copies / μL of single real-time fluorescent quantitative curve; b: M. DNA Marker 2000 Plus; 10 7 ~10 0 copies / μL of standard template conventional PCR amplification;-. negative control.

[0017] Figure 7 The results of the S. iniae sensitivity test. Among them, a: 1×10 0 ~1×10 7 copies / μL of single real-time fluorescent quantitative curve; b: M. DNA Marker 2000 Plus; 10 7 ~10 0 copies / μL of standard template conventional PCR amplification;-. negative control.

[0018] Figure 8 The results of the sensitivity test. Among them, green: YcCV TaqMan double real-time fluorescent quantitative PCR amplification curve; blue: S. iniae TaqMan double real-time fluorescent quantitative PCR amplification curve.

[0019] Figure 9 The standard curve. Among them, a: the standard curve of YcCV; b: the standard curve of S. iniae.

[0020] Figure 10 The freeze-thaw test.

[0021] Figure 11 The freeze-thaw test.

[0022] Figure 12 For the first day, store at -20℃, 4℃ and 28℃.

[0023] Figure 13 For the third day, store at -20℃, 4℃ and 28℃.

[0024] Figure 14 For the seventh day, store at -20℃, 4℃ and 28℃. DETAILED DESCRIPTION

[0025] The technical solutions described in the present application are conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or are already disclosed if not specifically stated.

[0026] By integrating molecular biology technology and epidemiological characteristic data, rapidly and sensitively detecting specific biomarkers in the early stage of pathogen infection or disease development will become a key path to break through the bottleneck of existing aquatic disease monitoring. By designing multiple probes targeting the conserved virulence genes of Streptococcus and the cupedovirus genes, homology interference can be effectively avoided and mutant strain recognition ability can be improved, thereby providing double protection for biological safety early warning of aquaculture ecosystems.

[0027] The embodiment of the present application provides a double detection primer and fluorescent probe for yellow catfish cupedovirus and dolphin Streptococcus, wherein the detection primer comprises primers shown as SEQ ID NO. 1-2 and SEQ ID NO. 4-5, and the fluorescent probe comprises fluorescent probes shown as SEQ ID NO. 3 and SEQ ID NO. 6.

[0028] In some specific embodiments, the 5' end of the fluorescent probe shown as SEQ ID NO. 3 is connected with a FAM group, and the 3' end is connected with a TAMRA group.

[0029] In some specific embodiments, the 5' end of the fluorescent probe shown as SEQ ID NO. 6 is connected with a HEX group, and the 3' end is connected with a BHQ1 group.

[0030] The embodiment of the present application also provides a kit for double detection of yellow catfish cupedovirus and dolphin Streptococcus, comprising the double detection primer and fluorescent probe for yellow catfish cupedovirus and dolphin Streptococcus.

[0031] The embodiment of the present application also provides a double detection method of yellow catfish cupedovirus and dolphin Streptococcus for non-disease diagnosis purposes, taking DNA of a sample to be tested as a template, performing real-time fluorescent quantitative reaction by using the double detection primer and fluorescent probe for yellow catfish cupedovirus and dolphin Streptococcus, and judging whether the sample contains yellow catfish cupedovirus and dolphin Streptococcus based on the Ct value of the template DNA.

[0032] In some specific embodiments, the reaction system of the real-time fluorescent quantitative reaction comprises: Premix ExTaq (Probe qPCR) (2X) 12.5 μL, S. iniae-Forward (10 μM) 0.5 μL, S. iniae-Reverse (10 μM) 0.5 μL, S. iniae-Probe (10 μM) 1.2 μL, YcCV-Forward (10 μM) 0.4 μL, YcCV-Reverse (10 μM) 0.4 μL, YcCV-Probe (10 μM) 1.2 μL, template 5 μL, and RNase Tree dH2O 3.3 μL.

[0033] In some specific embodiments, the reaction procedure of the real-time fluorescent quantitative reaction comprises: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, 45 cycles; 60°C for 30 s.

[0034] The embodiment of the present application also provides application of the double detection primer and fluorescent probe of the Pelteobagrus fulvidraco calicivirus and Streptococcus iniae in preparation of a product for detecting the Pelteobagrus fulvidraco calicivirus and Streptococcus iniae.

[0035] In some specific embodiments, the product comprises a drug, a reagent, or a kit.

[0036] Embodiment 1 1 Primer design The primer and probe of the aquatic animal-derived calicivirus (YcCV) are designed according to the YC-YcCV genome sequence (YC-25 Accession Number: MZ065194.1) in GenBank. The primer and probe for detecting Streptococcus iniae (S. iniae) are designed according to the Si-S. iniae genome sequence (SF1 Accession Number: CP005941.1) in GenBank.

[0037] Highly conserved fragments in ORF1 and attL1 genes are selected for analysis. The probe and primer (Table 1) designed for YcCV and S. iniae fragments are designed by using Primer Premier 6 software, and the primer design principle is referred to the Primer Express 3.0 guide of American Thermo (ABI) (Table 2). The primer and probe sequences designed in this section are synthesized by Shanghai Shengong Bioengineering Co., Ltd., and the purchased primer and probe are frozen at -20°C.

[0038] Table 1 Primer and probe sequences

[0039] Table 2 TaqMan fluorescent PCR probe and primer design guide

[0040] 2 Construction of positive plasmid The target sequence of this experiment was sent to Shanghai Shengong Bioengineering Co., Ltd. to construct the positive plasmids of YcCV and S. iniae, named as pUC57-YcCV and pUC57-S. iniae.

[0041] 3 TaqMan duplex fluorescent quantitative PCR amplification YcCV and S. iniae were amplified using specific primers in Table 1, with fragment lengths of 110 bp and 88 bp. The results showed that the length of the amplified target band was accurate, and the band was clear without impurity, consistent with the expected results, and the amplification results are shown in Figure 1 .

[0042] 3.1 Optimization of TaqMan duplex fluorescent quantitative PCR reaction conditions Judgment method: Premise: Design two probes (Table 1), FAM channel collects S. iniae fluorescence signal, HEX channel collects YcCV fluorescence signal. Control system meets the standard: no template control (NTC) has no obvious amplification curve in FAM and HEX channels, Ct value shows "Undetected" (not detected) or Ct value > 40; S. iniae positive control only FAM channel shows typical "S" shaped curve, Ct value is usually between 15-35, and the repeated experimental results are consistent; YcCV positive control only HEX channel shows typical "S" shaped curve, Ct value is usually between 15-35, and the repeated experimental results are consistent. If the control sample does not meet the above conditions, the reagent, contamination or instrument problem needs to be checked, and the result of the sample under test is invalid.

[0043] Specific judgment criteria: Only contains S. iniae: FAM channel (S. iniae): The amplification curve shows a typical "S" shape, and the Ct value is < 35; or 35 ≤ Ct ≤ 40, and there is still a clear "S" shaped curve after repeated detection; HEX channel (YcCV): No amplification curve, or Ct value > 40, no typical "S" shape growth.

[0044] Only contains YcCV: HEX channel (YcCV): The amplification curve shows a typical "S" shape, and the Ct value is < 35; or 35 ≤ Ct ≤ 40, and there is still a clear "S" shaped curve after repeated detection; FAM channel (S. iniae): No amplification curve, or Ct value > 40, no typical "S" shape growth.

[0045] Both viruses exist at the same time: FAM channel (S. iniae): meet the "only S. iniae" FAM channel determination condition; HEX channel (YcCV): meet the "only YcCV" HEX channel determination condition; the two-channel signal has no crosstalk, and the independent amplification curve of each channel has no mutual interference.

[0046] Both viruses do not exist: FAM channel (S. iniae): no amplification curve, or Ct value > 40, no typical "S" shape growth; HEX channel (YcCV): no amplification curve, or Ct value > 40, no typical "S" shape growth.

[0047] Taking pUC57-YcCV and pUC57-S. iniae as templates, under the conditions of TaqMan real-time fluorescence quantitative PCR reaction, a PCR reaction system with a total volume of 25 μL was established for optimization. 1 × 10 5 copies / μL of positive plasmid as template, sterile RNase-free water (DPEC) as negative control, without changing other conditions, changing a single variable, setting the annealing temperature to (57℃, 58℃, 59℃, 60℃), using a fluorescence quantitative PCR instrument to optimize the temperature range. Change the amount of primer and probe used, set the primer concentration and probe concentration to 100nM (0.2 μL), 150nM (0.3 μL), 200nM (0.4 μL), 250nM (0.5 μL), 300nM (0.6 μL) and 350nM (0.7 μL), respectively. The effect of primers and probes at different concentrations was tested by matrix method. According to the fluorescence intensity and the specific situation of the amplification curve, the results with smaller Ct value and maximum fluorescence intensity were selected to determine the best reaction system.

[0048] The optimal amount of primer was determined, and amplification reactions were performed with different primer concentrations. The primer concentration was adjusted to 100nM (0.2 μL), 150nM (0.3 μL), 200nM (0.4 μL), 250nM (0.5 μL), 300nM (0.6 μL) and 350nM (0.7 μL), respectively, and matrix method was used for screening. The experimental results showed that when the primer concentrations of S. iniae and YcCV were 250nM (0.5 μL) and 200nM (0.4 μL), respectively, the Ct value of the amplification reaction reached the lowest, which was 11.215 and 10.457, respectively (see Figure 2 ).

[0049] Effect of probe concentration on amplification reaction. The probe concentration was set at 300 nM (0.6 μL), 400 nM (0.8 μL), 500 nM (1 μL), 600 nM (1.2 μL), and 750 nM (1.5 μL), respectively. The experimental results showed that when the probe concentration of S. iniae and YcCV was 600 nM (1.2 μL), the Ct value of the amplification reaction reached the lowest, which was 11.301 and 10.637, respectively (see Figure 3 ).

[0050] By adjusting and optimizing the probe concentration of different primers, primer concentration and annealing temperature, since there was no obvious difference in the final results by comparison of different annealing temperatures, 60°C was selected as the best annealing temperature. In order to achieve the best effect, the best reaction system (Table 3) and reaction conditions (Table 4) of real-time fluorescent quantitative PCR were obtained. In the process of optimizing experimental parameters, the first consideration was to reduce the number of cycles while reducing non-specific amplification. When the experimental data was close, the primers that could significantly improve the fluorescence signal intensity and the probes that could work effectively at low concentration were selected to realize the efficiency and sensitivity of the experiment.

[0051] Table 3 qPCR reaction system

[0052] Table 4 qPCR reaction conditions

[0053] 3.2 TaqMan double fluorescent quantitative PCR specificity test Using virus DNA / RNA nucleic acid extraction kit to extract nucleic acid of Iridovirus (IV), Infectious Spleen and Kidney Necrosis Virus (ISKNV), Rhabdovirus (RV), and Infectious Pancreatic Necrosis Virus (IPNV) stored in the laboratory as template DNA, using positive plasmid of YcCV and S. iniae as positive nucleic acid, and using sterile RNAase-free water (DPEC) as negative control, PCR amplification was carried out under the optimized reaction conditions and system to test the specificity of primers and probes.

[0054] To evaluate the specificity of the established duplex TaqMan real-time PCR method for YcCV and S. iniae, several major fish viruses (IV, ISKNV, RV, IPNV and YcCV and S. iniae positive plasmid) were used as templates. YcCV and S. iniae positive plasmid was used as positive control, and sterile RNase-free water (DPEC) was used as negative control. The results showed that YcCV and S. iniae were detected positive (green, blue amplification curve), and no positive signal was detected for the other four fish viruses IV, ISKNV, RV, IPNV and negative control Figure 4 ), indicating that the established YcCV and S. iniae TaqMan RT-qPCR detection method has high specificity.

[0055] 3.3 Anti-interference of TaqMan duplex real-time fluorescence quantitative PCR Using standard RNA or DNA of major fish viruses, including IV, ISKNV, RV, IPNV and YcCV and S. iniae positive plasmid standard mixture as amplification template, sterile RNase-free water (DPEC) as negative control, the established duplex TaqMan real-time fluorescence quantitative PCR method was used for amplification.

[0056] To evaluate the anti-interference of the established duplex TaqMan real-time fluorescence quantitative PCR method for YcCV and S. iniae differential detection, several major fish viruses DNA / RNA and YcCV and S. iniae positive plasmid standard mixture used in the above experiment were used as amplification templates, and sterile RNase-free water (DPEC) was used as negative control. The results showed that YcCV and S. iniae were detected positive (green, blue amplification curve), indicating that the established YcCV and S. iniae duplex TaqMan real-time fluorescence quantitative PCR method has high anti-interference Figure 5 ).

[0057] 3.4 Establishment of TaqMan duplex real-time fluorescence quantitative PCR standard curve Using the optimized optimal reaction conditions, the positive plasmid standard was diluted by 10 times, and the concentration after dilution was used 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2Six dilutions of positive plasmids (copies / µL) were used as templates for Taqman dual real-time quantitative PCR. A blank control was set up using sterile RNase-free water (DPEC). Each PCR reaction and blank control were performed in triplicate. A standard curve was plotted using the results from the real-time quantitative PCR instrument.

[0058] YcCV and S. iniae positive plasmids were serially diluted 10-fold to 1.0 × 10⁻⁶. 2 copies / μL, at 1.0×10 7 1.0×10 6 1.0×10 5 1.0×10 4 1.0×10 3 1.0×10 2 The positive plasmid concentration was measured in copies / μL and amplified using the established dual TaqMan real-time quantitative PCR method. Standard curves for YcCV and S. iniae were plotted with the positive plasmid log concentration on the x-axis and Ct value on the y-axis. The standard curve equation for YcCV was y = -3.1648x + 35.018, with a correlation coefficient R² = 0.998; the standard curve equation for S. iniae was y = -3.1869x + 35.537, with a correlation coefficient R² = 0.998. (Standard curves...) Figure 9 They conform to a certain linear relationship.

[0059] 3.5 TaqMan Dual Real-Time PCR Sensitivity Assay To obtain the lowest detectable concentration of the positive plasmid, a serial dilution method was used, starting with sterile RNase-free water (DPEC) for serial dilution, and then continuously diluting the positive plasmid 8-fold, from 1.0 × 10⁻⁶. 7 Serial dilutions of copies / μL to 1.0×10⁻⁶ - 1 copies / µL, prepared into 1×10 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1 1×10 0 1×10 - 1 The positive plasmid concentration was determined by copies / µL, with DEPC water used as a negative control. TaqMan quantitative PCR and conventional PCR were performed to determine the minimum detection concentration.

[0060] Reaction condition of routine PCR: 95℃ 3min; 94℃ 25s; 60℃ annealing 25s, 35 cycles in total: 72℃ extension 10s. After the reaction, 6μL amplified product was electrophoresed in 2.0% agarose gel for 35min.

[0061] Reaction system of routine PCR: 7μL sterile RNase-free water, 10μL 2×M5 HiPer plus Taq Hifi PCR mix, 10μM upstream primer 0.5μL, 10μM downstream primer 0.5μL, 2μL DNA template.

[0062] To evaluate the sensitivity of the constructed duplex TaqMan real-time fluorescent quantitative PCR detection method, YcCV and S. iniae positive plasmid were used as templates for amplification reaction, Taqman single real-time fluorescent quantitative PCR reaction and routine PCR test were carried out respectively, and amplification curves were obtained (see Figure 6 、 7 for details). YcCV and S. iniae two positive plasmid mixture as template, TaqMan duplex real-time fluorescent quantitative PCR reaction was carried out, and amplification curve was obtained (see Figure 8 for details). In routine PCR reaction, the minimum detection limit of YcCV and S. iniae was 1×10 2 copies / μL and 1×10 2 copies / μL, respectively, while in Taqman single real-time fluorescent quantitative PCR reaction, the minimum detection limit of YcCV and S. iniae reached 1×10 0 copies / μL and 1×10 0 copies / μL, respectively. The results showed that the constructed duplex TaqMan real-time fluorescent quantitative PCR detection method had higher sensitivity.

[0063] 3.6 TaqMan duplex real-time fluorescent quantitative PCR repeatability test The template DNA of YcCV and S. iniae with copy number of 1.0×10 6 copies / μL, 1.0×10 5 copies / μL, 1.0×10 4 copies / μL was detected by Taqman duplex real-time fluorescent quantitative PCR. Intra-group repeatability test: at the same time, the same batch of reagents and consumables were used to detect three samples with three dilution degrees for three times; inter-group repeatability: the above three standard samples were tested for three times at different times under the same laboratory conditions. The coefficient of variation was calculated according to the Ct value.

[0064] To evaluate the repeatability of the duplex TaqMan real-time fluorescent quantitative PCR detection method that has been constructed, batch-in and batch-out repeated detection was performed. The standard template was diluted by 10 times successively, and the coefficient of variation CV of the duplex TaqMan real-time fluorescent quantitative PCR was detected. The results are shown in Table 5, and the variation coefficients of group-in and group-out repeatability are both less than 2%, indicating that the TaqMan duplex real-time fluorescent quantitative PCR detection method established in the present study has reliable repeatability.

[0065] Table 5 Results of repeatability test

[0066] 4 TaqMan duplex fluorescent quantitative PCR kit To preliminarily explore the stability of the main reaction system of the fish calicivirus and dolphin Streptococcus TaqMan duplex fluorescent quantitative PCR detection method established, the rapid detection kit includes qPCR reaction solution, enzyme mixture, positive control and blank control, and the experiment includes repeated freeze-thaw experiment and accelerated aging experiment. All components of the kit are operated by using a pipettor. The positive control is added in a biosafety cabinet in the CNAS laboratory million-level clean area. YcCV and S. iniae nucleic acids are detected in vitro. The detection principle and method are real-time fluorescent quantitative PCR.

[0067] 4.1 Composition of reaction system The kit mainly contains the following key components: qPCR reaction solution, enzyme mixture, positive control and blank control. The specific names, configuration specifications and core components of these components are shown in Table 6.

[0068] Table 6 Composition of kit

[0069] 4.2 Composition of qPCR reaction solution The composition of YcCV and S. iniae reaction solution is prepared in a 25 μL reaction system, and each box can react 50 times for detection. The composition and volume of the qPCR reaction solution of the fish calicivirus and dolphin Streptococcus TaqMan duplex fluorescent quantitative PCR detection kit are shown in Table 7.

[0070] Table 7 Composition and volume of qPCR reaction solution

[0071] 4.3 Stability evaluation of reconstitution of reaction system ​​​​​​​​​​​​​To explore whether the established detection method will be affected by the repeated freezing and thawing of the main reaction system. The qPCR reaction solution and enzyme mixture were mixed into the same batch of reaction solution, and the prepared main reaction system was repeatedly frozen and thawed at -80°C, and naturally thawed at room temperature of 25°C. The blank control and positive control were stored separately without freezing and thawing. Because the kit is designed for 50 portions per box, at least 3 portions are needed for one amplification reaction detection experiment, and at most 16 times of freezing and thawing per box, considering the freezing and thawing times during transportation, the freezing and thawing times of each well reaction were designed as 1 time, 3 times, 5 times, 7 times, 9 times, 12 times, 15 times, and 18 times. qPCR system amplification test was performed.

[0072] After the mixed reaction solution was repeatedly frozen and thawed at -80°C for 1 time, 3 times, 5 times, 7 times, 9 times, 12 times, 15 times, and 18 times, qPCR amplification detection was performed, and the amplification curve was obtained (see Figure 10 for details), and the fluorescence increase value did not change significantly with the increase of freezing and thawing times. The results showed that freezing and thawing for 18 times or less had no effect.

[0073] 4.4 Stability evaluation of positive control after repeated freezing and thawing To explore whether the established detection method will be affected by the repeated freezing and thawing of the main reaction system. The qPCR reaction solution and enzyme mixture were mixed into the same batch of reaction solution, and the prepared main reaction system was repeatedly frozen and thawed at -80°C, and naturally thawed at room temperature of 25°C. The blank control and positive control were stored separately without freezing and thawing. Because the kit is designed for 50 portions per box, at least 3 portions are needed for one amplification reaction detection experiment, and at most 16 times of freezing and thawing per box, considering the freezing and thawing times during transportation, the freezing and thawing times of each well reaction were designed as 1 time, 3 times, 5 times, 7 times, 9 times, 12 times, 15 times, and 18 times. qPCR system amplification test was performed.

[0074] After the positive control was repeatedly frozen and thawed at -80°C for 1 time, 3 times, 5 times, 7 times, 9 times, 12 times, 15 times, and 18 times, qPCR amplification detection was performed, and the amplification curve was obtained (see Figure 11 for details), and the fluorescence increase value did not change significantly with the increase of freezing and thawing times. The results showed that freezing and thawing for 18 times or less had no effect.

[0075] 4.5 Stability evaluation of short-term storage at -20°C, 4°C, and 28°C To explore whether the main reaction system of the established detection method will affect the detection effect under different storage times at-20℃, 4℃ and 28℃. The positive controls used in the experiment were high concentration, medium concentration and low concentration, each with 3 samples. The high concentration, medium concentration and low concentration samples were stored at-20℃, 4℃ and 28℃ for 1 day, 3 days and 7 days. The qPCR reaction solution and enzyme mixture in the kit were mixed according to the proportion to prepare the reaction mixture, and the total system was 25µL. The amplification was carried out according to the established TaqMan double fluorescent quantitative PCR detection method of fish calicivirus and dolphin streptococcus. The amplification results were used to judge whether the change of positive control storage time at-20℃, 4℃ and 28℃ would affect the detection effect.

[0076] In addition, the qPCR reaction solution and enzyme mixture were mixed according to the proportion to prepare the mixture, and each had 3 samples of high concentration, medium concentration and low concentration. The storage temperature and time were the same as above, and the experimental batch was consistent. The positive reference in the kit was used to prepare the reaction mixture according to the proportion, and the total system was 25µL. The amplification was carried out according to the established TaqMan double fluorescent quantitative PCR detection method of fish calicivirus and dolphin streptococcus. The amplification results were used to judge whether the change of qPCR reaction solution and enzyme mixture storage time at-20℃, 4℃ and 28℃ would affect the detection effect. The detection effect changes of the reaction solution with different storage times in the system under the same batch and the same time were compared.

[0077] 28℃ condition was used to simulate the storage stability evaluation under the condition of temperature loss during transportation. The kit usually needs to be stored and transported within a specific temperature range to maintain its activity and stability. The kit may be degraded during transportation due to temperature loss, so it is particularly important to determine the maximum storage time under specific transportation conditions. The reaction system configuration described above was placed in a constant temperature box at 28℃ to simulate the transportation conditions after the failure of the built-in ice bag under low temperature transportation conditions. The amplification results were used to judge whether the change of positive control storage time under different temperature loss conditions would affect the detection effect. The detection effect changes of the reaction solution with different storage times under the condition of temperature loss in the system under the same batch and the same time were compared.

[0078] Different concentrations of reaction solution were stored at-20℃, 4℃ and 28℃, and the same batch and the same time were compared under the condition of different storage times of reaction solution in the system. The storage condition at 28℃ can simulate the effective storage time of the reaction system during temperature loss transportation.

[0079] (1) On the first day of storage, the Ct values of the positive controls of different concentrations were as follows: Figure 12 The average Ct values of the detection were as shown in Tables 8 and 9.

[0080] Table 8 Ct values of positive controls of different concentrations stored for 1 day

[0081] Table 9 Ct values of different concentrations of reaction systems at 1 day storage

[0082] (2) At the 3rd day of storage, the average Ct values of the detection are as shown in Table 10, Table 11. Figure 13

[0083] Table 10 Ct values of different concentrations of positive controls at 3 days storage

[0084] Table 11 Ct values of different concentrations of reaction systems at 3 days storage

[0085] (3) At the 7th day of storage, the average Ct values of the detection are as shown in Table 12, Table 13. Figure 14

[0086] Table 12 Ct values of different concentrations of positive controls at 7 days storage

[0087] Table 13 Ct values of different concentrations of reaction systems at 7 days storage

[0088] Obviously, the above embodiments of the present application are merely exemplary and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. It is not necessary or possible to exhaust all the embodiments. Any modification, equivalent replacement and improvement within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.​​

Claims

1. A primer and fluorescent probe for the dual detection of yellow catfish calicivirus and dolphin streptococcus, characterized in that, The detection primers include those shown in SEQ ID NO.1-2 and SEQ ID NO.4-5, and the fluorescent probes include those shown in SEQ ID NO.3 and SEQ ID NO.

6.

2. The primers and fluorescent probes for the dual detection of yellow catfish calicivirus and dolphin streptococcus according to claim 1, characterized in that, The fluorescent probe shown in SEQ ID NO.3 has a 5' end linker of FAM and a 3' end linker of TAMRA.

3. The primers and fluorescent probes for the dual detection of yellow catfish calicivirus and dolphin streptococcus according to claim 1, characterized in that, The fluorescent probe shown in SEQ ID NO.6 has a 5' end linker HEX and a 3' end linker BHQ1.

4. A kit for dual detection of yellow catfish calicivirus and dolphin streptococcus, characterized in that, Includes the dual detection primers and fluorescent probes for yellow catfish calicivirus and dolphin streptococcus as described in any one of claims 1-3.

5. A dual detection method for yellow catfish calicivirus and dolphin streptococcus for non-disease diagnostic purposes, characterized in that, Using the DNA of the sample to be tested as a template, a real-time fluorescence quantitative reaction is performed using the dual detection primers and fluorescent probes for yellow catfish calicivirus and dolphin streptococcus as described in any one of claims 1-3. The presence of yellow catfish calicivirus and dolphin streptococcus is determined based on the Ct value of the template DNA.

6. The dual detection method as described in claim 5, characterized in that, The reaction system for the real-time quantitative PCR reaction includes: 12.5 μL of Premix Ex Taq (Probe qPCR) (2X), 0.5 μL of S. iniae-Forward (10 μM), 0.5 μL of S. iniae-Reverse (10 μM), 1.2 μL of S. iniae-Probe (10 μM), 0.4 μL of YcCV-Forward (10 μM), 0.4 μL of YcCV-Reverse (10 μM), 1.2 μL of YcCV-Probe (10 μM), 5 μL of template, and 3.3 μL of RNase Tree dH2O.

7. The dual detection method as described in claim 5, characterized in that, The reaction procedure for the real-time fluorescence quantitative reaction includes: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, 45 cycles; 60℃ for 30 s.

8. The use of the dual detection primers and fluorescent probes for yellow catfish calicivirus and dolphin streptococcus as described in any one of claims 1-3 in the preparation of products for detecting yellow catfish calicivirus and dolphin streptococcus.

9. The application as described in claim 8, characterized in that, The products include drugs, reagents, or kits.

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