Panda source rotavirus real-time fluorescence RT-RAA rapid detection method

Abstract

The invention is applicable to the technical field of virus detection, and provides a panda-source rotavirus real-time fluorescence RT-RAA rapid detection method, which specifically comprises the following steps: firstly, preparing a panda-source rotavirus RNA standard substance, then designing and synthesizing a specific primer and a probe aiming at a panda-source rotavirus VP7 gene, and finally detecting the panda-source rotavirus VP7 gene. The method comprises the following steps: firstly, extracting nucleic acid by using a virus DNA / RNA kit, then, establishing a real-time fluorescent RT-RAA detection method, then, carrying out sensitivity, specificity and repeatability tests, verifying the performance of the detection method, and finally, detecting a panda excrement sample. Under the condition that the temperature is 37 DEG C within 16 min, the lowest limit of detection concentration is 102 copies / reaction, virus RNA with extremely low concentration can be detected, the detection accuracy is improved, an amplification curve is generated only for rotavirus RNA, cross reaction with other viruses is avoided, the detection accuracy is ensured, intra-group and inter-group variable coefficients are lower than 10%, and the detection result is stable and reliable.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a real-time fluorescence RT-RAA rapid detection method for rotavirus from giant pandas. Background Art

[0002] Rotavirus (RV) belongs to the genus Rotavirus of the family Reoviridae. Its genome consists of linear double-stranded RNA, with a total gene length of about 18.525 kb. It is one of the main pathogens causing viral diarrhea in infants and young animals. Recombinase-Aided Amplification (RAA) is a newly emerging nucleic acid detection technology. Under constant temperature conditions, the recombinase extracted and purified from Escherichia coli forms a complex with two pairs of specific primers of the target gene. This complex binds tightly to the DNA strand. With the cooperation of single-stranded DNA binding protein (SSB), DNA unwinding is completed, and new DNA complementary strands are continuously formed under the action of DNA polymerase. By repeating this process, the reaction products increase exponentially and efficiently, ultimately achieving nucleic acid amplification. In 2016, Chen Zhenrong et al. detected the rotavirus carriage in 95 giant panda fecal samples by establishing fluorescence quantitative PCR. The results showed that 23 samples were positive, 1 sample was suspected to be positive, and the positive rate was 24.21%.

[0003] Adult animals infected with rotavirus mostly have a latent course and continuously excrete the virus to the outside world. The transmission routes are through the fecal-oral route and the water source route. Moreover, rotavirus has strong resistance to environmental factors and many common disinfectants. After an outbreak in a certain place, the area becomes an epidemic area, and the disease is constantly found throughout the year, which poses a major threat to the health, breeding, and reintroduction of giant pandas into the wild.

[0004] Therefore, in view of the above current situation, there is an urgent need to develop a real-time fluorescence RT-RAA rapid detection method for rotavirus from giant pandas to overcome the deficiencies in current practical applications. Summary of the Invention

[0005] The purpose of the embodiments of the present invention is to provide a real-time fluorescence RT-RAA rapid detection method for rotavirus from giant pandas, aiming to solve the problems raised in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A real-time fluorescence RT-RAA rapid detection method for rotavirus from giant pandas specifically includes the following steps:

[0008] S1: Preparation of the RNA standard of rotavirus from giant pandas;

[0009] S2: Design and synthesis of specific primers and probes for the VP7 gene of rotavirus from giant pandas;

[0010] S3: Extract nucleic acids using a viral DNA / RNA kit;

[0011] S4: Establish a real-time fluorescence RT-RAA detection method;

[0012] S5: Conduct sensitivity, specificity, and repeatability tests to verify the performance of the detection method;

[0013] S6: Detect giant panda fecal samples.

[0014] For a further technical solution, the preparation of the RNA standard in S1 includes the following steps: Select the rotavirus VP7 gene sequence, synthesize the RV-VP7 gene fragment using the RV-H gene sequence and clone it into the pVAX1 vector, and name the constructed recombinant plasmid pVAX1-RV-VP7; Use restriction enzymes to perform double digestion and linearization on the recombinant plasmid; Use an RNA synthesis kit to perform in vitro transcription on the digested product to obtain RNA; Use an RNA purification kit to purify the RNA, and use a UV spectrophotometer to measure the RNA concentration.

[0015] For a further technical solution, the design and synthesis of primers and probes in S2 includes the following steps: Download the full gene sequence of the VP7 gene of the RV-CH1 strain from the gene bank and use software for alignment; Select appropriate sequences from highly conserved regions to design multiple primers and probes.

[0016] For a further technical solution, use a DNA / RNA kit to extract the plasmids of canine distemper virus (CDV) from giant pandas, feline panleukopenia virus (FPV) from giant pandas, Schmallenberg virus (SBV), and the nucleic acids of peste des petits ruminants virus (PPRV), and store them at -80 °C.

[0017] For a further technical solution, the establishment of the real-time fluorescence RT-RAA detection method in S4 includes the following steps: Configure the reaction system including 29.4 μL of A Buffer, 8.5 μL of ddH2O, 2 μL of upstream and downstream primers, 0.6 μL of Probe, 2.5 μL of BBuffer, and 5 μL of RNA sample; Conduct preliminary experiments on conditions such as primer concentration, reaction temperature, and reaction time in the amplification system, and optimize by fixing a single factor variable; Take a 10-fold diluted RVRNA standard product and amplify it using the established real-time fluorescence RT-RAA method to establish a standard curve.

[0018] A further technical solution is that the sensitivity, specificity and repeatability test in S5 includes the following steps: the RNA standard is subjected to a 10-fold gradient release, and amplified using the established real-time fluorescence RT-RAA method to determine the sensitivity of the method; the nucleic acids of the common plague virus (CDV), feline panleukopenia virus (FPV), porcine reproductive and respiratory syndrome virus (SBV), and peste des petits ruminants virus (PPRV) are used as templates, and amplified using RT-RAA to evaluate the specificity of the method; RNA standards with three concentration gradients are taken respectively, and inter-group and intra-group repeated tests are carried out, with 3 replicates set for each concentration, and the coefficient of variation is calculated based on its Ct value to evaluate the repeatability of the method.

[0019] As a further technical solution, 10 negative healthy giant panda feces samples collected in S6 were artificially mixed with 4 RV plasmid standards with different copy numbers to simulate positive samples, and tested by real-time fluorescence RT-RAA experiment.

[0020] In summary, compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0021] 1. The detection method of the present invention can detect the lowest concentration of 10 within 16 minutes at 37°C. 2 The detection limit of real-time fluorescence RT-PCR is 10 0 Copy / reaction, which can detect extremely low concentrations of viral RNA and improve detection accuracy;

[0022] 2. Only produces an amplification curve for rotavirus RNA, with no cross-reaction with other viruses, ensuring detection accuracy;

[0023] 3. The coefficients of variation within and between groups were both lower than 10%, and the test results were stable and reliable.

[0024] In order to more clearly illustrate the structural features and effects of the present invention, the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the detection process of the present invention;

[0026] Figure 2 Schematic diagram of constructing the recombinant vector pVAX1-RV-VP7 in the present invention;

[0027] Figure 3 This is a schematic diagram of the plasmid electrophoresis results of the RNA standard in the present invention;

[0028] Figure 4 is a real-time fluorescence RT-RAA amplification curve diagram of different primer combinations in the present invention;

[0029] Figure 5 This is the real-time fluorescence RT-RAA amplification curve graph with a cycle time of 15 s in the present invention;

[0030] Figure 6 This is the real-time fluorescence RT-RAA amplification curve graph with an amplification temperature of 37 °C in the present invention;

[0031] Figure 7 This is the RV real-time fluorescence RT-RAA standard curve graph of the present invention;

[0032] Figure 8 This is the curve graph of the real-time fluorescence RT-PCR sensitivity test result of the present invention;

[0033] Figure 9 This is the curve graph of the real-time fluorescence RT-RAA sensitivity test result of the present invention;

[0034] Figure 10 This is the curve graph of the real-time fluorescence RT-RAA specificity test result of the present invention. Detailed implementation manners

[0035] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be 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.

[0036] The following describes the specific implementation of the present invention in detail in conjunction with specific embodiments.

[0037] As Figure 1 shown, the embodiment of the present invention provides a real-time fluorescence RT-RAA rapid detection method for rotavirus from giant pandas, which specifically includes the following steps:

[0038] S1: Preparation and identification of RNA standard products;

[0039] (1) Construction of recombinant plasmid: As Figure 2 shown, the VP7 gene sequence of rotavirus is selected, and the RV-VP7 gene fragment is synthesized using the RV-H gene sequence and cloned into the pVAX1 vector, and the constructed recombinant plasmid is named pVAX1-RV-VP7;

[0040] (2) Double digestion and linearization: The recombinant plasmid is double-digested using the restriction endonucleases HindIII-HF and BamHI, and then linearized using the restriction endonuclease SnaBI, and the digested products are recovered. Part of the digested products are used for in vitro transcription using an RNA synthesis kit to obtain RV RNA; in addition, part of the digested products are subjected to agarose gel electrophoresis for identification;

[0041] (3) RNA purification and concentration determination: Purify RNA using an RNA purification kit, measure the RNA concentration using an ultraviolet spectrophotometer, and calculate the copy number of in vitro transcribed RNA using the following formula.

[0042] RNA concentration (copies / μL) = [RNA concentration (ng / μL) × 10 -9 / (length of in vitro transcribed RNA × 340) × 6.02 × 10 23

[0043] Specifically, take 1 μg of DNA plasmid, 1 μL of restriction enzyme BamHI, 1 μL of restriction enzyme HindIII-HF, 5 μL of 10x NEBuffer, and make up to 50 μ

[0044] L with DNase / RNase-Free Water. Place it in a common PCR instrument and react at 37°C for 2 h. Then take the enzyme digestion product for 1.5% agarose gel electrophoresis. The results are as Figure 3 shown in A, presenting two bands. One is about 3000 bp and the other is about 1000 bp, which is consistent with the expected results. After linearizing pVAX1-RV-VP7 containing the RV-VP7 gene fragment with SnaBI digestion, take 2 μL of the enzyme digestion product, 1 μL each of the qPCR upstream and downstream primers, 12.5 μ

[0045] L of Taq man enzyme, and make up to 25 μL with DNase / RNase-Free Water. Put it into a common PCR instrument. The reaction program is 95°C for 2 min 30 s, 95°C for 30 s, 54°C for 30 s, 72°C for 1 min, for 35 cycles: 72°C for 10 min, 4°C

[0046] ∞. After the reaction, take 4 μL of the PCR product and 1 μL of 6x Loading buffer, mix well and use it for

[0047] 1.5% agarose gel electrophoresis. The results are as Figure 3 shown in B, presenting two bands, which is consistent with the expected results. After purifying the remaining enzyme digestion product, perform in vitro transcription using an RNA synthesis kit and purify it using an RNA purification kit to obtain the RVRNA standard. Subsequently, use an ultraviolet spectrophotometer to measure the RNA standard, and its concentration is 4.42 × 10 3 ng / μL, and calculate the copy number to be 7.46 × 10 12 copies / μL.

[0048] Figure 3 In A, double enzyme digestion is shown, and in B, single enzyme digestion is shown. 1 - 4 represent PCR products, 5 represents the single enzyme digestion recombinant plasmid, and 6 represents the non-enzyme digestion recombinant plasmid.

[0049] S2: Primer and probe design and synthesis;

[0050] Download the full gene sequence of the VP7 gene of the RV-CH1 strain from the gene bank, align it using SnapGene software, and select appropriate sequences from highly conserved regions to design multiple primers and probes. The following table shows the primers and probes for RV RT-RAA screening;

[0051]

[0052]

[0053] S3: Nucleic acid extraction;

[0054] Use a viral DNA / RNA kit to extract the nucleic acids of canine distemper virus (CDV) plasmid from giant pandas, feline panleukopenia virus (FPV) plasmid from giant pandas, Schmallenberg virus (SBV) plasmid, and peste des petits ruminants virus (PPRV), and store them at -80 °C for later use.

[0055] S4: Establish a real-time fluorescence RT-RAA detection method;

[0056] (1) Configure the reaction system: Use RT-fluorescent nucleic acid amplification reagent (RAA method) for the reaction. The reaction system is 50 μL, including 29.4 μL of A Buffer, 8.5 μL of ddH2O, 2 μL of upstream and downstream primers, 0.6 μL of Probe, 2.5 μL of BBuffer, and 5 μL of RNA sample;

[0057] (2) Optimize the reaction conditions: Conduct preliminary experiments on conditions such as primer concentration, reaction temperature, and reaction time in the amplification system, and optimize by fixing single-factor variables;

[0058] Specifically, for the 3 designed and synthesized forward primers and 2 reverse primers, combine them into 6 different primer pairs (F1-R1, F2-R1, F3-R1, F1-R2, F2-R2, F3-R2), and select 10 6 , 10 5 , 10 4 copies of the RNA standard as the template for real-time fluorescence RAA amplification. Select the optimal primer by analyzing the starting time of the logarithmic growth of the fluorescence signal. The reaction temperatures for isothermal amplification are set at 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, and 42 °C respectively to determine the optimal reaction temperature. The cycle times are set at 10 s, 15 s, 20 s, 25 s, 30 s, and 35 s respectively, and the total amplification time range is 10 min - 35 min. Determine the optimal reaction time based on the results of the amplification efficiency;

[0059] Such asFigure 4 As shown, the primer combination F3-R2 has the shortest slope starting time for amplifying the target fragment and the highest fluorescence value at the end. Therefore, F3-R2 was selected for the real-time fluorescence RT-RAA detection of RV;

[0060] As Figure 5 and Figure 6 shown, using the determined optimal primer F3-R2 and the same batch of RNA standard as the template, fluorescence RT-RAA amplification reactions were carried out under different amplification temperatures (37°C, 38°C, 39°C, 40°C, 41°C, 42°C) and different cycle times (10 s, 15 s, 20 s, 25 s, 30 s, 35 s). The results showed that positive detection lines could be observed in the above tests. The amplification efficiency was the highest at an amplification temperature of 37°C and a cycle time of 15 s. Considering factors such as time efficiency, fluorescence value, and amplification curve, the optimal reaction temperature and cycle time for the real-time fluorescence RT-RAA of RV were finally determined to be 37°C and 15 s, respectively.

[0061] (3) Construction of the standard curve: Take the 10-fold diluted RV RNA standard (1×10 5 copies / μL to 1×10 0 copies / μL), and perform amplification using the established real-time fluorescence RT-RAA method for RV to establish the standard curve.

[0062] In specific applications, the RV RNA standard of 1×10 5 copies / μL to 1×10 0 copies / μL was subjected to real-time fluorescence RT-RAA reaction according to the optimized reaction conditions, and repeated measurements were performed 3 times. At the same time, real-time fluorescence RT-PCR was used as a parallel control experiment to establish the standard curve. The results were as Figure 7 shown. There was a correlation between the Ct value of the real-time fluorescence RT-RAA reaction of RV and the logarithm of the copy number. The correlation coefficient R 2 was 0.951, the slope was -4.7874, and the regression equation was: y = -4.7874x + 36.964.

[0063] For real-time fluorescence RT-PCR, the following reaction solution (for one reaction) was prepared on ice. The reaction system is as shown in the following table. The reaction conditions were: 42°C for 5 min, 95°C for 10 sec, 1 Cycle. 95°C for 5 sec, 60°C for 20 sec, 40 Cycles. 95°C for 0 sec, 65°C for 15 sec, 95°C for 0 sec. Nucleic acid amplification was carried out on a fluorescence quantitative PCR instrument.

[0064]

[0065]

[0066] S5: Sensitivity, specificity, and repeatability tests;

[0067] (1) Sensitivity test: The RNA standard was serially diluted 10-fold to concentrations ranging from 1×10 5 copies / μL to 1×10 0 copies / μL. The established RV real-time fluorescence RT-RAA method was used for amplification to determine the sensitivity of the method. ddH2O was used as a negative control, and each experiment was repeated 6 times.

[0068] In specific applications, the RNA standard was diluted 10-fold (1×10 5 copies / μL to 1×10 0 copies / μL), and this was used as a template for the sensitivity detection of the RV real-time fluorescence RT-RAA method. The results are as Figure 8 shown; under the condition of 37°C within 16 min, the lowest detection limit of the concentration by this method was 10 2 copies / reaction (amplification time threshold = CT value × 0.5), while the lowest detection limit detected by the real-time fluorescence RT-PCR method was 10 0 copies / reaction. The results are as Figure 9 shown.

[0069] (2) Specificity test: Nucleic acids of canine distemper virus (CDV), feline panleukopenia virus (FPV), porcine reproductive and respiratory syndrome virus (SBV), and peste des petits ruminants virus (PPRV) were used as templates respectively, RV RNA was used as a positive control, and ddH2O was used as a blank control. RT-RAA was used for amplification to evaluate the specificity of the method.

[0070] In specific applications, the established real-time fluorescence RT-RAA method was used to amplify RV RNA standard and nucleic acids of FPV, PPRV, CDV, and SBV respectively. The results are as Figure 10 shown, where 1 represents RV, 2 represents FPV, 3 represents CDV, 4 represents SBV, 5 represents PPRV, and 6 represents the negative control. Only RV showed an amplification curve, and no amplification occurred for the nucleic acids of the remaining viruses and the negative control, indicating that this method can specifically detect RV RNA and does not cross-react with FPV, PPRV, CDV, and SBV.

[0071] (3) Repeatability test: RNA standards at three concentration gradients (1×10 6 copies / μL, 1×10 4 copies / μL, 1×10 2copies / μL), perform repeated tests between and within groups. Set 3 replicates for each concentration, calculate the coefficient of variation based on their Ct values, and evaluate the repeatability of this method.

[0072] In specific applications, as shown in the following table, by performing within-group and between-group repeatability tests on 3 RNA standard products with different concentrations, analyze the within-group and between-group coefficients of variation (CV%). The results show that the coefficient of variation of within-group repeats is 7.8% - 9.2%, and the coefficient of variation of between-group repeats is 3.9% - 9.4%. The CV values within and between groups are both below 10%, indicating that this method has high repeatability.

[0073]

[0074] S6: Sample detection;

[0075] Take 10 fecal samples from negative healthy giant pandas, artificially add RV plasmid standards with four different copy numbers (1×10 7 copies / μL, 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL) to simulate positive samples, and at the same time set up a positive control (RNA standard) and a negative control (ddH2O) to perform real-time fluorescence RT-RAA experiments. At the same time, use real-time fluorescence RT-PCR method for detection, compare the detection results of the two, and calculate the compliance of the two methods.

[0076] In specific applications, the RT-RAA detection method is used to detect 10 simulated samples (4 positive and 6 negative), and at the same time compare with the results of the RV fluorescence RT-qPCR test. The results are shown in the following table. The established real-time fluorescence RT-RAA method detects 4 positive samples (CT < 35) and 6 negative samples (CT ≥ 35 or no curve amplification), which is consistent with the results of 4 positive (CT < 35) and 6 negative results (CT ≥ 35 or no curve amplification) detected by RV RT-qPCR; compared with RV RT-qPCR, the diagnostic sensitivity and specificity of the RV real-time fluorescence RT-RAA method are 100%.

[0077]

[0078] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A real-time fluorescence RT-RAA rapid detection method for giant panda-derived rotavirus, characterized in that: The specific steps include: S1: Preparation of giant panda-derived rotavirus RNA standards; S2: Design and synthesize primers and probes specific to the VP7 gene of rotavirus from giant pandas; S3: Extract nucleic acid using a viral DNA / RNA kit; S4: Establish real-time fluorescence RT-RAA detection method; S5: Conduct sensitivity, specificity and repeatability tests to verify the performance of the detection method; S6: Test the panda feces samples.

2. The method for rapid detection of giant panda-derived rotavirus by real-time fluorescence RT-RAA according to claim 1, characterized in that: The preparation of RNA standards in S1 includes the following steps: The rotavirus VP7 gene sequence was selected, and the RV-VP7 gene fragment was synthesized using the RV-H gene sequence and cloned into the pVAX1 vector to construct a recombinant plasmid named pVAX1-RV-VP7; The recombinant plasmid was double-digested and linearized using restriction endonucleases; RNA was obtained by in vitro transcription of the enzyme digestion product using an RNA synthesis kit; Purify RNA using an RNA purification kit and measure the RNA concentration using a UV spectrophotometer.

3. The real-time fluorescence RT-RAA rapid detection method of giant panda-derived rotavirus according to claim 1, characterized in that: The design and synthesis of primers and probes in S2 include the following steps: The full gene sequence of the VP7 gene of the RV-CH1 strain was downloaded from the gene bank and compared using software; Appropriate sequences were selected from highly conserved regions to design multiple primers and probes.

4. The method for rapid detection of giant panda-derived rotavirus by real-time fluorescence RT-RAA according to claim 1, characterized in that: In S3, the nucleic acids of giant panda-derived canine distemper virus (CDV) plasmid, giant panda-derived feline panleukopenia virus (FPV) plasmid, Schmallenberg virus (SBV) plasmid, and peste des petits ruminants virus (PPRV) were extracted using a DNA / RNA kit and stored at -80°C.

5. The method for rapid detection of giant panda-derived rotavirus by real-time fluorescence RT-RAA according to claim 1, characterized in that: The establishment of the real-time fluorescence RT-RAA detection method in S4 includes the following steps: The reaction system was configured to include 29.4 μL of A Buffer, 8.5 μL of ddH2O, 2 μL of upstream and downstream primers, 0.6 μL of Probe, 2.5 μL of B Buffer, and 5 μL of RNA sample; Conduct preliminary experiments on the primer concentration, reaction temperature, reaction time and other conditions in the amplification system, and optimize them by using fixed single-factor variables; A 10-fold diluted RVRNA standard was taken and amplified using the established real-time fluorescence RT-RAA method to establish a standard curve.

6. The method for rapid detection of giant panda-derived rotavirus by real-time fluorescence RT-RAA according to claim 1, characterized in that: The sensitivity, specificity, and repeatability tests in S5 include the following steps: The RNA standards were diluted in a 10-fold gradient and amplified using the established real-time fluorescence RT-RAA method to determine the sensitivity of the method; The nucleic acids of CDV, FPV, SBV and PPRV were used as templates to amplify the DNA of peste des petits ruminants virus (PPRV) using RT-RAA to evaluate the specificity of the method. Three concentration gradients of RNA standards were taken for inter-group and intra-group repeated tests. Three replicates were set for each concentration. The coefficient of variation was calculated based on its Ct value to evaluate the repeatability of the method.

7. The method for rapid detection of giant panda-derived rotavirus by real-time fluorescence RT-RAA according to claim 1, characterized in that: In S6, 10 negative healthy giant panda fecal samples were collected and artificially mixed with 4 RV plasmid standards with different copy numbers to simulate positive samples, and detected by real-time fluorescence RT-RAA experiment.

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