Primer probe group, kit and method for detecting porcine rotavirus based on RT-RAA-LFD technology

Abstract

The invention belongs to the technical field of biology, and particularly relates to a primer probe group, a kit and a method for detecting porcine rotavirus (PoRV) based on an RT-RAA-LFD technology. According to the invention, a specific primer probe group is designed according to a PoRV VP6 gene conserved sequence, an amplification system is constructed and optimized, and a method for rapidly detecting PoRV based on an RT-RAA-LFD technology is established. According to the method, a reaction is carried out at the constant temperature of 37 DEG C for 15 min, amplification of PoRV nucleic acid can be achieved, the lowest detection limit is 22.3 copies / microliter, cross reaction with other common viral diarrhea viruses is avoided, the total coincidence rate of RT-RAA-LFD and RT-PCR detection methods is 97%, and the Kappa coefficient is 0.93 (K is larger than 0.75). The RT-RAA-LFD detection method established by the invention has good specificity, sensitivity and stability, has the characteristics of high detection speed, visualization, suitability for on-site rapid detection and the like, and provides a new technical means for rapid diagnosis and epidemiological investigation of PoRV in clinical culture or field operation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a primer probe set, a kit and a method for detecting porcine rotavirus based on RT-RAA-LFD technology. Background Art

[0002] Porcine rotavirus (PoRV) is one of the main viral pathogens that causes diarrhea in pigs. Clinically, it manifests as diarrhea, lethargy, rapid weight loss in piglets, and yellow or gray feces. Infection in fattening pigs can lead to slow growth and reduced feed conversion rate. Rotavirus can be divided into 10 different groups (RVA-RVJ) based on the VP6 nucleotide sequence, among which rotavirus group A (RVA), rotavirus group B (RVB), rotavirus group C (RVC), rotavirus group E (RVE) and rotavirus group H (RVH) have been detected in pigs. The current prevalent rotavirus is mainly the RVA group, and RVB and RVC groups have also been reported. The RVA group can be further divided into G1-G6, G8-G12 and G26 genotypes based on the VP7 gene. At present, the main rotaviruses prevalent in my country are G4, G5, G7 and G9, and the existing rotavirus vaccine is G5.

[0003] Porcine rotavirus is often co-infected with enteroviruses such as PEDV, TGEV, and PDCoV, and the clinical symptoms are very similar. It is urgent to establish a simple and rapid diagnostic method. At present, etiology, serology, and molecular biology detection are the main diagnostic methods for PoRV, but they have disadvantages such as cumbersome operation, time-consuming and costly, long test cycle, and high requirements for operators, which have certain limitations in clinical rapid detection applications. Therefore, the establishment of an efficient and convenient porcine rotavirus diagnostic method is conducive to the diagnosis and prevention of the disease. Recombinase polymerase isothermal amplification (RAA) is a new type of nucleic acid detection method with independent intellectual property rights in my country. It has the advantages of rapid response, simple operation, and support for diversified data reading

[11] . It does not require special instruments and professional laboratories, is easier to apply in clinical practice, and has strong promotion. Combining RAA with lateral flow dipstick (LFD) can realize the visualization of detection results, which is particularly suitable for field detection or clinical rapid detection. This study established a rapid detection method for PoRV using RT-RAA-LFD technology, aiming to provide a fast and simple technical means for frontline clinical diagnosis and epidemic investigation. Summary of the invention

[0004] The object of the present invention is to provide a primer probe set, a kit and a method for detecting porcine rotavirus based on RT-RAA-LFD technology.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A primer probe set for detecting porcine rotavirus based on RT-RAA-LFD technology, wherein the nucleotide sequence of the primer probe set is: Primer PoRV-F2: 5'-TTGAAAATGCTAAAACTACCATTGAATACTTT-3', Primer PoRV-R1: 5'-AAATTCCAATTCTCAATGTAATCAGAT-3', Probe PoRV-Probe: 5'-FAM-GGATGAAATAGCCAGAGAATCACAACGAAAT-THF-TGATAGCTCCACAAT-C3-SPACER-3'.

[0006] The use of the above primer probe set in preparing an RT-RAA-LFD kit for detecting porcine rotavirus.

[0007] An RT-RAA-LFD kit for detecting porcine rotavirus, comprising the above-mentioned primer probe set; The working concentrations of primer PoRV-F2, primer PoRV-R1 and probe PoRV-Probe in the kit are all 1.0 μM; The RT-RAA-LFD amplification reaction procedure of the kit is: react at 37°C for 15 minutes.

[0008] A method for detecting porcine rotavirus based on RT-RAA-LFD technology, the method is for non-disease diagnosis and treatment purposes, using RNA extracted from a sample to be tested as a template, using the above-mentioned primer probe set to perform RT-RAA-LFD amplification to obtain an amplified product, and using a test strip to visually interpret the amplified product; The working concentrations of the primer PoRV-F2, primer PoRV-R1 and probe PoRV-Probe in the primer-probe set are all 1.0 μM; The reaction procedure of the RT-RAA-LFD amplification is as follows: react at 37°C for 15 min; The result judgment criteria are: a positive result has two red stripes, one on the test line and the other on the quality control line; a negative result has only one red stripe on the quality control line and no red stripe on the test line; if only a red stripe appears on the test line and no red stripe appears on the quality control line, it is an invalid test result.

[0009] The significant advantages of the present invention are: The present invention designs a specific primer probe set based on the conservative sequence of the PoRVVP6 gene, constructs and optimizes the amplification system, and establishes a method for rapid detection of PoRV based on RT-RAA-LFD technology. This method can achieve the amplification of PoRV nucleic acid by constant temperature reaction at 37°C for 15 minutes, with a minimum detection limit of 22.3 copies / μL, and no cross-reaction with other common viral diarrhea viruses. The total consistency rate of RT-RAA-LFD and RT-PCR detection methods is 97%, and the Kappa coefficient is 0.93 (K﹥0.75). The RT-RAA-LFD detection method established by the present invention has good specificity, sensitivity and stability, and has the characteristics of fast detection speed, visualization and suitability for rapid on-site detection, etc., which provides a new technical means for rapid diagnosis and epidemiological investigation of PoRV in breeding clinics or field operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 :RT-RAA-LFD primer screening results.

[0011] Figure 2 : RT-RAA-LFD primer working concentration screening results.

[0012] Figure 3 : RT-RAA-LFD probe working concentration screening results.

[0013] Figure 4 :RT-RAA-LFD amplification reaction temperature screening results.

[0014] Figure 5 :RT-RAA-LFD amplification reaction time screening results.

[0015] Figure 6 : RT-RAA-LFD specificity test results.

[0016] Figure 7 : Results of RT-RAA-LFD sensitivity test.

[0017] Figure 8 : RT-RAA-LFD repeatability test results. DETAILED DESCRIPTION

[0018] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0019] Example 1 1 Materials and methods 1.1 Main reagents, nucleic acids and samples The viral RNA nucleic acid extraction kit was purchased from Hangzhou Biori Technology Co., Ltd. RT-RAA-nfo nucleic acid amplification kit (strip type, S006ZC) and disposable lateral flow chromatography test strip (R103ZC) were purchased from Hangzhou Zhongce Biotechnology Co., Ltd. The pUC57-VP6 plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd., which was obtained by connecting the porcine rotavirus VP6 gene shown in SEQ ID NO.1 to the pUC57 plasmid. Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), porcine deltacoronavirus (PDCoV), and porcine teschovirus (PTV) positive nucleic acids were preserved by the laboratory. 100 clinical diarrhea samples were obtained from large-scale pig farms in Fujian Province and stored at -20°C.

[0020] 1.2 Primer design and synthesis The porcine rotavirus VP6 gene sequences (JQ343834.1, MZ165494.1, etc.) were downloaded from GenBank, and the conserved regions were selected by NCBIBLAST alignment. RAA primers and nfo probes were designed using Oligo 7.0 and Primer 5.0 (Table 1). The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0021] Table 1PoRVRT-RAA-LFD primers and probes 1.3 RT-RAA-LFD primer screening The designed two upstream primers and three downstream primers were combined in pairs to obtain six primer pair combinations (Table 1). According to the instructions of the RT-RAA-nfo nucleic acid amplification reagent, a 50 μL reaction system was established: 1 dry powder reaction tube, 25.0 μL of buffer A, 2.0 μL of upstream primer (2 μM) and downstream primer (2 μM), 0.6 μL of probe (2 μM), 12.9 μL of ddH2O, 5.0 μL of pUC57-VP6 plasmid and 2.5 μL of buffer B. According to the reaction system, first mix the buffer A Buffer, upstream primer, downstream primer, probe, ddH2O, and RNA sample of each group evenly and add them to the dry powder reaction tube, then add the buffer B Buffer to the detection unit tube cap, cover the tube cap, mix thoroughly and centrifuge at low speed for 5 seconds, put the above reaction tube into the PCR instrument and incubate at 37℃ for 30 minutes. After the reaction product is diluted 6 times, it is detected using a disposable lateral flow chromatography test strip and the result is determined within 10 minutes. The best primer pair is screened out through the test results.

[0022] 1.4 Optimization of RT-RAA-LFD reaction system The screened best primers were diluted so that their final concentrations in the reaction system were 0.1 μM, 0.5 μM, 1.0 μM, 2.0 μM, and 5.0 μM. The above 50.0 μL reaction system was used to amplify the best primer working concentration by RT-RAA-LFD and the test strip results were used to screen out the best primer working concentration; based on the best primers, the probes were diluted so that their final concentrations in the reaction system were 0.1 μM, 0.5 μM, 1.0 μM, 2.0 μM, and 5.0 μM. The above 50.0 μL reaction system was used to amplify the best probe working concentration by RT-RAA-LFD and the test strip results were used to screen out the best probe working concentration; based on the best primers and probe working concentrations, the reaction temperatures were set to 35°C, 37°C, 39°C, 41°C, 43°C, and 45°C for RT-RAA-LFD reaction to screen out the best reaction temperature; finally, the reaction times were set to 1 min, 5 min, 10 min, 15 min, 20 min, and 25 min, respectively, to optimize the best reaction time.

[0023] 1.5RT-RAA-LFD specificity test used RNA of PEDV, TGEV, PoRV, and PDCoV as templates and ddH2O as negative control. RT-RAA-LFD amplification was performed under the optimized optimal reaction conditions and detected using disposable lateral flow chromatography test strips. The specificity of the method was evaluated by the test results.

[0024] 1.6 RT-RAA-LFD sensitivity test The concentration of pUC57-VP6 plasmid was determined by NanoDrop2000, and the copy number was calculated based on the plasmid concentration. The copy number (copies / μL) = 6.02 × 10 23 × concentration (ng / μL) × 10 -9 / (number of bases × 660), the copy number of the recombinant plasmid pUC57-VP6 was 2.23 × 10 10 The recombinant plasmid pUC57-VP6 was diluted 10-fold to obtain 2.23-2.23×10 4 ddH2O was used as negative control and five working concentrations were used as templates for RT-RAA-LFD amplification. The detection was performed using disposable lateral flow chromatography test strips. The sensitivity of the method was evaluated by the test results.

[0025] 1.7RT-RAA-LFD repeatability test Take pUC57-VP6 plasmid and dilute it to a concentration of 2.23×10 1 copies / μL, 2.23×10 2copies / μL, 2.23×10 3 The repeatability experiment was carried out 3 times for 100 copies / μL respectively, and the disposable lateral flow chromatography test strips were used for detection. The repeatability of the method was evaluated by the test results.

[0026] 1.8 Clinical sample testing RNA was extracted from 100 clinical diarrhea samples or anal swabs, and the samples were tested using the RT-RAA-LFD method established in this study. The samples were compared with the RT-PCR method (SN / T 5196-2020). The positive rates of the two detection methods were analyzed and compared, and the conformity of the two detection methods was calculated to evaluate the practical application effect of the detection method.

[0027] 2 Results 2.1 RT-RAA-LFD Primer Screening RT-RAA-LFD amplification was performed according to the primer combinations (F1 / R1, F1 / R2, F1 / R3, F2 / R1, F2 / R2, F2 / R3), and the primer pairs were screened based on the test results. The results showed that ( Figure 1 ): F1 / R1, F1 / R2 and F2 / R1 can amplify pUC57-VP6 plasmid, while F1 / R3, F2 / R2 and F2 / R3 cannot be used as primers due to false positives. Among them, F2 / R1 has the shortest amplified fragment, the best amplification efficiency and the best test strip brightness. F2 / R1 was selected as the best primer for the next step of reaction system optimization and specificity, sensitivity and repeatability experiments.

[0028] 2.2 Optimization of RT-RAA-LFD reaction conditions The working concentrations of primers, probes, RT-RAA-LFD amplification reaction temperature and reaction time were optimized. The optimization results are as follows: Figures 2 to 5 shown.

[0029] The band brightness of the T line of the test strip increased with the increase of the primer working concentration when the primer working concentration was 0.1μM~1.0μM. The band brightness was the highest when the primer working concentration was 1.0μM, but the higher working concentrations of 2.0μM and 5.0μM primers did not increase the band brightness of the T line ( Figure 2 ). Therefore, the optimal reaction concentration of upstream and downstream primers is 1.0 μM.

[0030] The brightness of the T line of the test strip cannot produce a detection reaction when the probe working concentration is 0.1μM. When the probe working concentration is 0.5-1.0μM, it increases with the concentration. When the probe working concentration is increased to 2.0μM, the band cannot increase the brightness of the T line, and when the probe working concentration is 5.0μM, the brightness of the T line is reduced ( Figure 3 ). Therefore, the optimal reaction probe working concentration is 1.0 μM.

[0031] The brightness of the T line of the test strip increases with the temperature when the reaction temperature is 35℃~37℃. When the reaction temperature is increased to 39℃~43℃, the brightness of the T line cannot be improved. When the temperature is 45℃, the T line cannot produce a detection reaction ( Figure 4 ). Therefore, considering the clinical field operating environment, 37°C was selected as the optimal reaction temperature.

[0032] The brightness of the T line of the test strip cannot produce a detection reaction when the reaction time is 5 minutes. When the reaction time is 10 minutes, 15 minutes and 20 minutes, the brightness of the T line of the test strip increases with the increase of reaction time and reaches the brightest at 15 minutes. However, there is no significant difference in the brightness of the T line of the test strip when the reaction time is 20 minutes and 25 minutes. Figure 5 ). Therefore, in order to save detection time, the screening reaction time was 15 min.

[0033] 2.2 Specificity test The positive nucleic acids of PEDV, TGEV, PoRV, PDCoV and PTV were used as templates, and ddH2O was used as a negative control. The optimized reaction system was used for RT-RAA-LFD detection; namely, the RT-RAA-LFD amplification reaction system: 1 dry powder reaction tube, 25.0 μL of buffer A, 2.0 μL of upstream primer F2 (1.0 μM) and downstream primer R1 (1.0 μM), 0.6 μL of probe (1.0 μM), 12.9 μL of ddH2O, 5.0 μL of template and 2.5 μL of buffer B. The RT-RAA-LFD amplification reaction conditions were: 37°C for 15 min. The results showed ( Figure 6 ), except for the reaction product with PoRV as template which can be detected on the T test line of the test strip, other viral diarrhea pathogens related to diarrhea were detected as negative by RT-RAA-LFD. This shows that the RT-RAA-LFD detection method established in this study has good specificity and is not interfered by other common viral diarrhea pathogens.

[0034] 2.3 Sensitivity test The pUC57-VP6 plasmid was diluted 10-fold to 2.23-2.23×10 4 5 working concentrations of copies / μL were used as templates, ddH2O was used as negative control template, and RT-RAA-LFD detection was performed using the optimized reaction system. The results showed that ( Figure 7 ), as the template concentration decreases, the brightness of the test strip T detection line decreases until 2.23×10 1A weak detection line can be seen at 10 copies / μL, and when the template concentration is 2.23×10 0 copies / μL could not be detected, indicating that the minimum detection limit of RT-RAA-LFD of the present invention was 2.23×10 1 copies / μL.

[0035] 2.3 Repeatability test With a concentration of 2.23×10 1 copies / μL, 2.23×10 2 The pUC57-VP6 plasmid with 100 copies / μL was used as the template, and the experiment was repeated three times. The optimized reaction system was used for RT-RAA-LFD detection. The results showed that ( Figure 8 ), at two different concentrations, the RT-RAA-LFD method can accurately detect, and the brightness of the T line of the test strip is consistent. This shows that the RT-RAA-LFD detection method established by the present invention has good repeatability.

[0036] 2.3 Clinical sample testing The extracted diarrhea sample or anal swab RNA was used as a template, and the RT-RAA-LFD method and RT-PCR method established and optimized by the present invention were used for detection, and the results showed (Table 2) that the RT-RAA-LFD method detected 34 (34 / 100) positive samples, with a positive rate of 34.0%, and the RT-PCR method detected 32 (32 / 100) positive samples, with a positive rate of 32.0%. Compared with the two methods, 2 samples were detected as positive by the RT-RAA-LFD method, while the RT-PCR method detected them as negative, and 1 sample was detected as positive by the RT-PCR method, while the RT-RAA-LFD method detected them as negative. The positive predictive value of the RT-RAA-LFD method was 96.88%, the negative predictive value was 98.51%, the specificity was 97.06%, the sensitivity was 96.88%, the total compliance rate was 97.00%, and the Kappa coefficient was 0.93 (K>0.75). The results showed that the RT-RAA-LFD method was highly consistent with the RT-PCR method and had higher detection accuracy.

[0037] Table 2 Clinical sample test results Note: TP: true positive, TN: true negative, FP: false positive, FT: false negative. Positive predictive value = TP / (TP+FP); Negative predictive value = TN / (TN+FT); Specificity = TN / (TN+FP); Sensitivity = TP / (TP+FT); Total coincidence rate = (TP+TN) / total number of samples; The larger the Kappa (K) value, the better the consistency; K<0.4 indicates low consistency, and K≥0.75 indicates high consistency.

[0038] PoRV is one of the most important viral diarrhea pathogens in farms, which seriously endangers the development of pig farming. Although RT-PCR, qPCR, LAMP and other detection methods for PoRV have been reported, they require specific instruments and operators, which limits their use outside laboratory conditions. The RAA combined with lateral flow chromatography test strip detection method has low requirements for the detection environment and equipment, is easy to operate, and can obtain results within 5 to 15 minutes, making the detection results visual, which has promotion value. The RT-RAA-LFD detection method established in this study can complete amplification at 37°C for 15 minutes, with a minimum detection limit of 22.3 copies / μL. The sensitivity is higher than that of the reported PoRV SYBR Green I RT-qPCR detection method (Wu Yang. Establishment and preliminary application of multiple and SYBR GreenⅠ fluorescence quantitative PCR detection methods for porcine diarrhea-related viruses [D]. Northeast Agricultural University, 2014), and the PoRV TaqMan The sensitivity of RT-qPCR is comparable to that of RT-qPCR (Lin Zhengdan, Tu Jun, Zhan Cunlin, et al. Establishment and preliminary application of TaqMan fluorescence quantitative detection method for porcine rotavirus [J]. Chinese Journal of Veterinary Medicine, 2024, 44(02): 244-248.; Liu Jiajia, Liang Haiying, Zeng Zhiyong, et al. Establishment and application of TaqMan real-time fluorescence quantitative RT-PCR detection method for porcine rotavirus [J]. Chinese Journal of Veterinary Science, 2024, 54(02): 162-168.); this method does not cross-react with other common diarrheal viral pathogens, and repeatability tests can be well amplified, indicating that it has good specificity and repeatability. This method was used to test 100 clinical samples. The RT-RAA-LFD results showed that the PoRV positivity rate was 34.00% (34 / 1003), while the RT-qPCR method (SN / T 5196-2020) positivity rate was 32.00% (32 / 100). Although the test results of the two were not completely consistent, the consistency rate between the two was 97.00%, and the Kappa coefficient was 0.93 (K﹥0.75), which showed that the RAA-LFD method had a higher detection accuracy.

[0039] The amplification primers and probes of RAA are the key to establishing the method. Since there is no specific software design, the following principles should be met when designing primers: the probe is labeled with a fluorescent group (FAM), and the 5' end of the primer opposite to the probe sequence is labeled with biotin, or the probe is labeled with biotin and the primer is labeled with a fluorescent group; the probe length is preferably 46-52 bp, of which 30 nt is located at the 5' end of the tetrahydrofuran (THF) position, and at least 15 bp is located at the 3' end of the THF position; THF replaces a base between 30 bp downstream of the fluorescent group and 15 bp upstream of the blocking group; the blocking group C3-Spacer needs to be added to the 3' end of the probe. In this study, two upstream primers and three downstream primers were designed for pairwise combination, and six sets of primer pairs were obtained for amplification experiments. It was found that the amplified fragment had a better amplification effect at 188 bp, indicating that when designing primers for the RAA amplification method, attention should be paid to controlling the amplified fragment within 200 bp. When screening the concentration of probes and primers, it was found that the optimal reaction concentration was 1.0 μM, while high concentrations of probes and primers not only failed to improve the amplification efficiency, but had a negative effect, indicating that different primer concentrations had a certain impact on the amplification of RAA and the visualization of LFD. At the same time, although the RRA-LFD technology is easy to operate, can achieve rapid on-site detection without instruments, is cost-effective, and has advantages in remote areas with limited resources, this method has high requirements for primer design, which is prone to false positives.

[0040] The PoRV visual RAA detection method established in this study is simple to operate, quick to respond, highly specific, and highly sensitive. It can realize the visual judgment of the test results with the naked eye and is suitable for on-site detection of PoRV in clinical frontline conditions in the field, providing a new technical means for the diagnosis, epidemiological investigation, and prevention and control of PoRV.

[0041] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A primer probe set for detecting porcine rotavirus based on RT-RAA-LFD technology, characterized in that: The nucleotide sequence of the primer probe set is: Primer PoRV-F2: 5'-TTGAAAATGCTAAAACTACCATTGAATACTTT-3', Primer PoRV-R1: 5'-AAATTCCAATTCTCAATGTAATCAGAT-3', Probe PoRV-Probe: 5'-FAM-GGATGAAATAGCCAGAGAATCACAACGAAAT-THF-TGATAGCTCCACAAT-C3-SPACER-3'.

2. Use of the primer probe set according to claim 1 in preparing an RT-RAA-LFD kit for detecting porcine rotavirus.

3. An RT-RAA-LFD kit for detecting porcine rotavirus, characterized in that: Comprising the primer probe set according to claim 1.

4. The kit according to claim 3, characterized in that: The working concentrations of the primer PoRV-F2, the primer PoRV-R1 and the probe PoRV-Probe in the kit are all 1.0 μM.

5. The kit according to claim 3, characterized in that: The RT-RAA-LFD amplification reaction conditions of the kit are: 37° C. for 15 min.

6. A method for detecting porcine rotavirus based on RT-RAA-LFD technology, the method is for non-disease diagnosis and treatment purposes, characterized in that: The RNA extracted from the sample to be tested is used as a template, and the primer probe set according to claim 1 is used to perform RT-RAA-LFD amplification to obtain an amplified product, and a test strip is used to visually interpret the amplified product.

7. The method according to claim 6, characterized in that: The working concentrations of the primer PoRV-F2, the primer PoRV-R1 and the probe PoRV-Probe in the primer-probe set are all 1.0 μM.

8. The method according to claim 6, characterized in that: The reaction conditions of the RT-RAA-LFD amplification are: 37° C. for 15 min.

Images