Fluorescent PCR primer-probe set, kit and method for simultaneously detecting GAstV and RA

Abstract

The present invention discloses a fluorescence PCR primer-probe set, kit and method for simultaneously detecting GAstV and RA. The primer-probe set includes 4 different primers and 2 different probes. Among them, 2 primers for amplifying GAstV have nucleotide sequences as shown in SEQ ID NO.3 to 4; 1 probe for amplifying GAstV has a nucleic acid sequence as shown in SEQ ID NO.17; 2 primers for amplifying RA have nucleotide sequences as shown in SEQ ID NO.11 to 12; 1 probe for amplifying RA has a nucleotide sequence as shown in SEQ ID NO.18. It can be used for simultaneously detecting GAstV and RA, and has advantages such as simple operation, high sensitivity, good specificity, fast detection speed and low cost. It is suitable for large-scale clinical promotion and application, and has good promotion value and application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology detection, and particularly relates to a fluorescence PCR primer and probe set, a kit and a method for simultaneously detecting GAstV and RA. Background Art

[0002] Goose astrovirus (GAstV) is a member of the genus Avastrovirus in the family Astroviridae. It has no envelope, the virus particles have a diameter of 28 - 30 nm, show an icosahedral structure, and the genome is single-stranded positive-sense RNA, with a size of approximately 6.1 - 7.9 kb. GAstV is the pathogen causing gosling gout, which is a newly emerging disease in goslings in recent years. Clinically, it is mainly manifested as listlessness, reduced food intake, poor growth and development, excretion of lime-like loose stools, emaciation, dehydration, and paralysis. At autopsy, a large amount of urate deposition can be seen in internal organs and joints. All breeds of geese can be infected, and the incidence rate is high in goslings aged 8 - 19 days. The incidence rate of goslings infected with it is over 80%, and the mortality rate is 30% - 50%. In 2016, this disease was first reported in China and then spread rapidly across the country. Currently, it has been reported in major goose-producing areas such as Jiangsu, Shandong, Anhui, Guangdong, and Henan in China, causing serious economic losses to the goose farming industry in China. <![CDATA[ ]]<![CDATA[]]><![CDATA[

[0003] ]]<![CDATA[]]><0000><![CDATA

[033] ]><![CDATA[]]><![CDATA[

[0003] ]]<![CDATA[]]>Riemerella anatipestifer (RV) is a Gram-negative bacterium that can infect waterfowl such as ducks and geese, causing Riemerella anatipestifer, also known as infectious serositis. Goslings within 2 months of age are most severely affected, with an incidence rate of over 90% and a mortality rate as high as 75%, causing serious economic losses to goose farming. In 1932, this disease was first reported in the United States and in China in 1982. The clinical manifestations of Riemerella anatipestifer disease in geese are mainly neurological symptoms, fibrinous pericarditis, perihepatitis, and air sac inflammation, which are similar to duck Escherichia coli disease and duck Salmonella disease. It is very difficult to make a differential diagnosis clinically and is one of the most serious diseases endangering the goose farming industry.

[0004] Both GAstV and RA are among the most serious diseases currently harming the goose - raising industry. The onset age of goslings is similar, which can cause a large number of gosling deaths, resulting in serious losses. Moreover, it is very difficult to diagnose solely based on clinical symptoms, and laboratory detection techniques are needed. Traditional detection methods for GAstV and RA include pathogen isolation, fluorescent antibody technique, slide agglutination test, enzyme - linked immunosorbent assay (ELISA), PCR method, etc. However, each of these detection methods has its own disadvantages, such as being time - consuming and laborious, prone to cross - reactions, having low detection sensitivity, or having high requirements for instruments, equipment, and personnel. Fluorescent PCR technology has been widely used in the detection of various diseases and is currently the best choice for diagnosing these two diseases. Although there are currently single - item fluorescent PCR methods for GAstV or RA respectively, there is no dual - fluorescent PCR method for simultaneously detecting GAstV and RA. A technology for simultaneously and rapidly detecting GAstV and RA is urgently needed, and a kit suitable for on - site rapid detection, high - efficiency, sensitivity, and simplicity needs to be developed. Primers and probes are the most critical components for kit preparation. Currently, the multiplex fluorescent PCR detection technology is not yet mature, and it is not easy to design primers and probes for multiple fluorescent PCR detections. Interference and matching between different primers and probes need to be considered, and the ratio of each primer and probe needs to be continuously optimized, not a simple and random ratio. To achieve the best amplification effect, the annealing temperature and reaction conditions of the amplification reaction also need to be continuously optimized to determine the optimal primer - probe ratio and reaction conditions. Therefore, various conditions need to be optimized during the establishment of the fluorescent PCR method. Reagents from different manufacturers are selected for comparison and analysis to select reagents with high cost - performance and good detection effects. The dual - fluorescent PCR kit for simultaneously detecting GAstV and RA of the present invention can fill the blank in the related field. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a fluorescent PCR primer - probe set for simultaneously detecting GAstV and RA; the second objective of the present invention is to provide a kit containing the above - mentioned fluorescent PCR primer - probe set for simultaneously detecting GAstV and RA; the third objective of the present invention is to provide a method for detecting GAstV and RA using the above - mentioned kit.

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

[0007] 1. A fluorescent PCR primer - probe set for simultaneously detecting GAstV and RA

[0008] It includes 4 different primers and 2 different probes. Among them, there are 2 primers for amplifying GAstV, and their nucleotide sequences are shown as SEQ ID NO.3-4; there is 1 probe for amplifying GAstV, and its nucleic acid sequence is shown as SEQ ID NO.17; there are 2 primers for amplifying RA, and their nucleotide sequences are shown as SEQ ID NO.11-12; there is 1 probe for amplifying RA, and its nucleotide sequence is shown as SEQ ID NO.18.

[0009] Preferably in the present invention, the 5′ end of the sequence of the probe for amplifying GAstV is labeled with the fluorescent group FAM, and the 3′ end is labeled with the quenching group BHQ1.

[0010] Preferably in the present invention, the 5′ end of the sequence of the probe for amplifying RA is labeled with the fluorescent group HEX, and the 3′ end is labeled with the quenching group BHQ1.

[0011] 2. A kit containing the above-mentioned fluorescent PCR primer-probe set for simultaneously detecting GAstV and RA, which includes the following reagents:

[0012] (1) PCR reaction solution;

[0013] (2) DNA polymerase, reverse transcriptase, dNTP mixture;

[0014] (3) Primer and probe premixed solution: a mixed solution of the primers shown in SEQ ID NO.3-4, SEQ ID NO.11-12 and the probes shown in SEQ ID NO.17-18;

[0015] Preferably in the present invention, the kit further includes the following reagents:

[0016] (4) Positive control: an equal-volume mixed solution of the recombinant plasmid pMD-3′UTR containing the GAstV gene and the recombinant plasmid pMD-pyrC containing the RA gene;

[0017] (5) Negative control: purified water.

[0018] Preferably in the present invention, in the primer and probe premixed solution, the molar ratio of the primers shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.11 and SEQ ID NO.12 is 8:12:7:11, and the molar ratio of the probes shown in SEQ ID NO.17 and SEQ ID NO.18 is 5:4.

[0019] Preferably in the present invention, the preparation concentrations of the primers shown in SEQ ID NO.3-~4 and SEQ ID NO.11-12 are both 15 μmol / L, and the preparation concentrations of the probes shown in SEQ ID NO.17-18 are both 10 μmol / L.

[0020] Preferably, in the positive control, the concentration of plasmid pMD-3′UTR is 2.8×10 3 copies / μL, and the concentration of RA plasmid pMD-pyrC is 4.3×10 3 copies / μL.

[0021] 3. A method for detecting GAstV and RA using the kit, comprising the following steps:

[0022] Mix the test sample with the PCR reaction solution, DNA polymerase, reverse transcriptase, dNTP mixture, primer and probe premix in the kit, and perform fluorescence PCR reaction according to the following reaction program: reverse transcription at 42°C for 5 min; pre-denaturation at 95°C for 10 s, denaturation at 95°C for 5 s, annealing at 55°C for 20 s (collect fluorescence here), for a total of 40 cycles.

[0023] The beneficial effects of the present invention are as follows: For the GAstV and RA gene sequences, multiple sets of primers and probes are designed respectively, the best combination is screened out, and a kit is assembled based on the primer-probe group. The ratios of each primer and probe and the amplification reaction conditions are optimized. Through the change of fluorescence signal, the nucleic acids of two pathogens, GAstV and RA, can be detected simultaneously. The kit is easy to operate, reduces the risk of laboratory aerosol contamination, has good stability, realizes rapid, simple and high-throughput detection of GAstV and RA, and has the advantages of good specificity, high sensitivity, accurate results and high-throughput detection. It is suitable for large-scale popularization and application, and has high commercial value and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0025] Figure 1 Sensitivity test for GAstV detection by dual fluorescence PCR method;

[0026] Figure 2 Sensitivity test for RA detection by dual fluorescence PCR method;

[0027] Figure 3 Specificity test for GAstV detection by dual fluorescence PCR method;

[0028] Figure 4 Specificity test for RA detection by dual fluorescence PCR method. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited do not limit the present invention.

[0030] Example 1

[0031] Screening and comparative verification of fluorescence PCR primer pairs

[0032] Four groups of primers were designed for the GAstV 3′UTR gene (accession number: OM273303.1) and the RA pyrC gene sequence (accession number: LT906475.1), and combined with GAstV and RA specific probes (SEQ ID NO.17, SEQ ID NO.18, the probes for GAstV and RA corresponding to the four groups of primers are the same).

[0033] The probe sequences are as follows:

[0034] GAstV-P: 5’-ACAGCTGCACCTTCTCATTAGGCTTGAG-3’ (SEQ ID NO.17);

[0035] RA-P: 5’-TATAGAACAGCCCAACACCGTTCCTAATG-3’ (SEQ ID NO.18);

[0036] The specific nucleotides of primer group A are as follows:

[0037] GAstV-F1: 5’-ATCTCTTCACTAGCAGCCGC-3’ (SEQ ID NO.1);

[0038] GAstV-R1: 5’-CTGTCACCGGGTTGATTTA-3’ (SEQ ID NO.2);

[0039] RA-F1: 5’-ACTATTGAAACAGAAAGCCG-3’ (SEQ ID NO.9);

[0040] RA-R1: 5’-CTATCTGATATTTATTTTCTA-3’ (SEQ ID NO.10);

[0041] The specific nucleotides of primer group B are as follows:

[0042] GAstV-F2: 5’-GCCACGCCGAGTAGGAT-3’ (SEQ ID NO.3);

[0043] GAstV-R2: 5'-GGTTGATTTAAGGCAGAG-3' (SEQ ID NO.4);

[0044] RA-F2: 5'-GTTGCTGGAGGTGTTA-3' (SEQ ID NO.11);

[0045] RA-R2: 5'-GAGTAGTTGGCGTGGG-3' (SEQ ID NO.12);

[0046] The specific nucleotides of the primers in Group C are as follows:

[0047] GAstV-F3: 5'-CTAGCAGCCGCGGCCACGC-3' (SEQ ID NO.5);

[0048] GAstV-R3: 5'-TAAGGCAGAGAGGGCCGATTC-3' (SEQ ID NO.6);

[0049] RA-F3: 5'-TTACCCATAAAGGAACTATT-3' (SEQ ID NO.13);

[0050] RA-R3: 5'-ATTTATTTTCTAAAAGTTC-3' (SEQ ID NO.14);

[0051] The specific nucleotides of the primers in Group D are as follows:

[0052] GAstV-F4: 5'-CGCCGAGTAGGATCGAGG-3' (SEQ ID NO.7);

[0053] GAstV-R4: 5'-ACGATTTGTGTGGGTGAC-3' (SEQ ID NO.8);

[0054] RA-F4: 5'-GCCGTGCTGCCGTTGCTG-3' (SEQ ID NO.15);

[0055] RA-R4: 5'-GTGGGAAGTTTGCGAAGC-3' (SEQ ID NO.16);

[0056] The above primers and probes were synthesized by General Biosystems (Anhui) Co., Ltd.

[0057] When the primers in each group and the two probes were mixed and applied in a molar ratio of 1:1:1:1:1:1, with the annealing temperature of 53°C and the template fixed, fluorescence PCR amplification reactions were carried out. The results are shown in Table 1:

[0058] Table 1. Screening of optimal primer sets

[0059] Primer group Primer group A Primer group B Primer group C Primer group D GAstV amplification result CT22.5 CT21.5 CT23.2 No amplification RA amplification result CT21.1 CT20.6 No amplification CT20.9

[0060] The results show that group B is the best primer group screened by the present invention. The amplification effects of the other primer groups are not ideal. Among them, the primers in group C RA did not amplify, the primers in group D GAstV did not amplify, and the primers in group A were not sensitive enough and there were cases of missed detection.

[0061] Example 2

[0062] Establishment of a fluorescent PCR method for simultaneous detection of GAstV and RA

[0063] 1. Optimal Primer Probe Set

[0064] The GAstV 3′UTR gene and RA pyrC gene sequences were retrieved and downloaded from GenBank. After comparison and analysis using DNAMAN software, multiple sets of primers and probes were designed. The optimal primer and probe set sequences obtained after screening are as follows:

[0065] The primers used for GAstV amplification were:

[0066] Forward primer SEQ ID NO. 3: 5′-GCCACGCCGAGTAGGAT-3′;

[0067] Reverse primer SEQ ID NO. 4: 5′-GGTTGATTTAAGGCAGAG-3′;

[0068] The primers used for RA amplification were:

[0069] Forward primer SEQ ID NO. 11: 5′-GTTGCTGGAGGTGTTA-3′;

[0070] Reverse primer SEQ ID NO. 12: 5′-GAGTAGTTGGCGTGGG-3′;

[0071] The probes used for GAstV amplification are:

[0072] SEQ ID NO.17: 5'-ACAGCTGCACCTTCTCATTAGGCTTGAG-3';

[0073] The probes used for RA amplification are:

[0074] SEQ ID NO.18: 5'-TATAGAACAGCCCAACACCGTTCCTAATG-3';

[0075] The 5′ end of the probe shown in SEQ ID NO.17 is labeled with the fluorescent group FAM, and the 3′ end is labeled with the quenching group BHQ1. The 5′ end of the probe shown in SEQ ID NO.18 is labeled with the fluorescent group HEX, and the 3′ end is labeled with the quenching group BHQ1.

[0076] II. Preparation of positive control

[0077] In this example, the positive control is an equal - volume mixture of the recombinant plasmid pMD - 3′UTR containing the GAstV 3′UTR gene and the recombinant plasmid pMD - pyrC containing the RA pyrC gene. Before mixing, the concentration of plasmid pMD - 3′UTR is 5.6×10 3 copies / μL, and the concentration of plasmid pMD - pyrC is 8.6×10 3 copies / μL.

[0078] III. Optimization of reaction system and procedure

[0079] Take the best - screened primer set B. The prepared concentrations of the primers shown in SEQ ID NO.3 - 4 and SEQ ID NO.11 - 12 are both 15 μmol / L, and the prepared concentrations of the probes shown in SEQ ID NO.17 - 18 are both 10 μmol / L. With different annealing temperatures of 51℃, 53℃, 55℃, 57℃, 59℃, 61℃, 62℃, keep the template unchanged. Then, keep the annealing temperature fixed, and mix each primer and probe in the same volume for fluorescence PCR amplification reaction. The optimization results of the annealing temperature are shown in Table 2:

[0080] Table 2 Optimization of annealing temperature

[0081] Annealing temperature 51℃ 53℃ 55℃ 57℃ 59℃ 61℃ 62℃ GAstV amplification result CT21.6 CT22.3 CT21.2 CT22.5 CT26.8 CT26.7 CT28.4 RA amplification result CT24.3 CT23.7 CT20.8 CT24.8 CT25.6 CT26.7 CT26.5

[0082] Take the best - screened primer set B. The prepared concentrations of the primers shown in SEQ ID NO.3 - 4 and SEQ ID NO.11 - 12 are both 15 μmol / L, and the prepared concentrations of the probes shown in SEQ ID NO.17 - 18 are both 10 μmol / L. When mixing each primer and probe of GAstV and RA in different volumes, with an annealing temperature of 55℃ and keeping the template unchanged, perform fluorescence PCR amplification reaction. The results are shown in Table 3:

[0083] Table 3 Optimization of primer concentration

[0084] Primer-probe volume ratio 1.1:1.3:0.8:0.9:0.5:0.8 0.8:1.2:0.5:0.7:1.1:0.4 1:1:1:1:1:1:1:1 1:1.5:1.2:0.5:1.6:0.9 GAstV amplification result CT24.5 CT22.7 CT23.8 CT26.5 RA amplification result CT25.2 CT23.6 CT24.9 CT25.7

[0085] Note: The volume ratio of primers and probes is SEQ ID NO.3:SEQ ID NO.4:SEQ ID NO.17:SEQ ID NO.11:SEQ ID NO.12:SEQ ID NO.18.

[0086] Finally, the total reaction system was determined to be 20 μL: 10 μL of 2×One Step RT-PCR Buffer, 0.8 μL of the mixed solution of Ex Taq HS and PrimeScript RT Enzyme Mix in a volume ratio of 1:1, 4.7 μL of the primer-probe mixed solution, 2.5 μL of purified water, and 2 μL of the template were mixed well, totaling 20 μL. Among them, the primer-probe mixed solution contained 0.8 μL of SEQ ID NO.3 (15 μmol / L), 1.2 μL of SEQ ID NO.4 (15 μmol / L), 0.5 μL of SEQ ID NO.17 (10 μmol / L), 0.7 μL of SEQ ID NO.11 (15 μmol / L), 1.1 μL of SEQ ID NO.12 (15 μmol / L), and 0.4 μL of SEQ ID NO.18 (10 μmol / L). The optimized best reaction program was: reverse transcription at 42°C for 5 min; pre-denaturation at 95°C for 10 s, denaturation at 95°C for 5 s, annealing at 55°C for 20 s (fluorescence was collected here), for a total of 40 cycles.

[0087] IV. Result determination

[0088] After the fluorescence PCR reaction ended, the software of the real-time fluorescence PCR instrument was used to analyze the test samples based on the amplification curves of the real-time fluorescence PCR. The result determination criteria for analyzing the test samples were as follows:

[0089] Quality control criteria:

[0090] For the positive control, the Ct values of both the FAM and HEX channels should be ≤ 30.0, and a typical "S"-shaped amplification curve should appear; for the negative control, there should be no Ct values in both the FAM and HEX channels, and no typical amplification curve. The test can be determined to be valid only when both the positive control and the negative control are established; otherwise, the test is invalid.

[0091] Result description and determination:

[0092] If the Ct value of the test sample in the FAM channel ≤ 35.0 and a typical "S"-shaped amplification curve appears, it is determined as positive for GAstV nucleic acid; if the test sample has no Ct value in the FAM channel and no typical amplification curve, it is determined as negative for GAstV nucleic acid; if the Ct value of the test sample is 35.0 < Ct value ≤ 40.0 and a typical "S"-shaped amplification curve appears, it is determined as suspicious. The suspicious samples are subjected to a double-well repeat test. If the result of any one well or both wells in the repeat test is positive, it is determined as positive for GAstV nucleic acid, otherwise it is determined as negative for GAstV nucleic acid.

[0093] If the Ct value of the test sample in the HEX channel ≤ 35.0 and a typical "S"-shaped amplification curve appears, it is determined as positive for RA nucleic acid; if the test sample has no Ct value in the HEX channel and no typical amplification curve, it is determined as negative for RA nucleic acid; if the Ct value of the test sample is 35.0 < Ct value ≤ 40.0 and a typical "S"-shaped amplification curve appears, it is determined as suspicious. The suspicious samples are subjected to a double-well repeat test. If the result of any one well or both wells in the repeat test is positive, it is determined as positive for RA nucleic acid, otherwise it is determined as negative for RA nucleic acid.

[0094] Example 3

[0095] Sensitivity test of the fluorescence PCR kit for simultaneous detection of GAstV and RA

[0096] The positive control plasmids pMD-3′UTR and pMD-pyrC were serially diluted 10-fold respectively. Take the plasmid standard pMD-3′UTR with a concentration of 5.6×10 2 copies / μL to 5.6×10 -2 copies / μL, and the plasmid standard pMD-pyrC with a concentration of 8.6×10 2 copies / μL to 8.6×10 -2 copies / μL as templates, and perform duplex fluorescence PCR detection using the kit prepared with the primer-probe set described in Example 2. The detection system and detection conditions are as described in Example 2.

[0097] The results are as Figure 1 and Figure 2 shown, Figure 1 In it, 1 represents the plasmid standard pMD-3′UTR of 5.6×10 2 copies / μL; 2 represents the plasmid standard pMD-3′UTR of 5.6×10 1 copies / μL; 3 represents the plasmid standard pMD-3′UTR of 5.6×10 0 copies / μL; 4 represents the plasmid standard pMD-3′UTR of 5.6×10 -1 copies / μL; 5 represents the plasmid standard pMD-3′UTR of 5.6×10-2 pMD - 3′UTR plasmid standard at copies / μL; 6 represents the negative control.

[0098] Figure 2 In 1 represents 8.6×10 2 pMD - pyrC plasmid standard at copies / μL; 2 represents 8.6×10 1 pMD - pyrC plasmid standard at copies / μL; 3 represents 8.6×10 0 pMD - pyrC plasmid standard at copies / μL; 4 represents 8.6×10 - 1 pMD - pyrC plasmid standard at copies / μL; 5 represents 8.6×10 -2 pMD - pyrC plasmid standard at copies / μL; 6 represents the negative control.

[0099] The detection results show that the minimum detection limit of the duplex fluorescence PCR method for GAstV is 0.56 copies / μL, and the minimum detection limit for RA is 0.86 copies / μL. The results indicate that the multiplex fluorescence quantitative PCR method established in the present invention has high sensitivity.

[0100] Example 4

[0101] Specificity test of the fluorescence PCR kit for simultaneous detection of GAstV and RA

[0102] Take 6 different pathogens such as avian influenza virus, goose paramyxovirus, goose circovirus, goose parvovirus, goose Escherichia coli, and goose Salmonella. After extracting nucleic acids respectively as templates, use the developed fluorescence PCR kit for amplification detection. At the same time, set positive controls of GAstV or RA, and a negative control of purified water.

[0103] The results are as Figure 3 (Results of the FAM fluorescence channel) and Figure 4 (Results of the HEX fluorescence channel) shown. In Figure 3 1 represents GAstV; 2 represents RA; 3 represents avian influenza virus; 4 represents goose paramyxovirus; 5 represents goose circovirus; 6 represents goose parvovirus; 7 represents goose Escherichia coli; 8 represents goose Salmonella; 9 represents purified water.

[0104] In Figure 4 1 represents RA; 2 represents GAstV; 3 represents avian influenza virus; 4 represents goose paramyxovirus; 5 represents goose circovirus; 6 represents goose parvovirus; 7 represents goose Escherichia coli; 8 represents goose Salmonella; 9 represents purified water.

[0105] GAstV has a typical S-shaped amplification curve in the FAM fluorescence channel, and RA has a typical S-shaped amplification curve in the HEX fluorescence channel, and the CT values of both are ≤ 30.0. There are no amplification curves for the remaining samples and the negative control, indicating that the method of the present invention has good specificity.

[0106] Example 5

[0107] Repeatability test of the fluorescence PCR kit for simultaneous detection of GAstV and RA

[0108] Take the plasmid standard pMD-3′UTR with a concentration of 5.6×10 6 copies / μL, serially diluted 10-fold. Take 5.6×10 4 copies / μL, 5.6×10 3 copies / μL, and 5.6×10 2 copies / μL of the three different dilutions for within-batch and between-batch repeatability tests.

[0109] Take the plasmid standard pMD-pyrC with a concentration of 5.6×10 6 copies / μL, serially diluted 10-fold. Take 8.6×10 4 copies / μL, 8.6×10 3 copies / μL, and 8.6×10 2 copies / μL of the three different dilutions for within-batch and between-batch repeatability tests.

[0110] Within-batch repeatability test: Set 3 replicates for each sample and detect with the prepared fluorescence PCR kit under the same test. Between-batch repeatability test: Conduct 3 independent detections with the developed fluorescence PCR kit under the same test conditions at different time periods. Calculate the standard deviation and coefficient of variation based on the Ct values to verify the repeatability of the method. The results are shown in Table 4. The within-batch and between-batch coefficients of variation of plasmid pMD-3′UTR and pMD-pyrC are both not higher than 2%, indicating that the kit has good repeatability.

[0111] Table 4 Results of the repeatability test of the fluorescence quantitative PCR detection kit

[0112]

[0113] Example 6

[0114] Detection of clinical samples using the kit of the present invention

[0115] Collect 120 suspected goose liver samples infected with GAstV or RA, process them by conventional methods, add physiological saline at a ratio of 1:10, stir and mix evenly, and extract total nucleic acid as a template. Detect using the fluorescence PCR kit for simultaneous detection of GAstV and RA of the present invention, and at the same time detect using the GAstV fluorescence PCR detection method specified in the literature "Establishment and Preliminary Application of TaqMan Fluorescence Quantitative PCR Detection Method for Goose-origin Astrovirus" and the RA fluorescence PCR detection method specified in the literature "Establishment of TaqMan Fluorescence Quantitative PCR Detection Method for Riemerella anatipestifer", and compare the detection results of the kit of the present invention with the reference method. The results show that the kit of the present invention detected 18 GAstV-positive samples and 21 RA-positive samples, while the reference method detected 16 GAstV-positive samples and 18 RA-positive samples. It can be seen that the kit of the present invention has higher sensitivity compared with the detection methods in the references and has good commercial application prospects.

[0116] As can be seen from the above embodiments, the present invention provides a primer-probe set and develops a fluorescence PCR kit for simultaneous detection of GAstV and RA based on the primer-probe set. This kit is different from the kits currently on the market and can simultaneously detect the nucleic acids of two pathogens, GAstV and RA. The kit has strong anti-interference ability, is easy to operate, has high sensitivity and good specificity, does not require a professional PCR laboratory, and can be used in conjunction with a portable fluorescence detection device to achieve rapid on-site detection of pathogens, with high commercial value and good application prospects.

[0117] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. A fluorescence PCR primer-probe set for simultaneously detecting GAstV and RA, characterized in that, It includes 4 different primers and 2 different probes. Among them, 2 primers for amplifying GAstV have nucleotide sequences shown in SEQ ID NO.3 - 4; 1 probe for detecting GAstV has a nucleic acid sequence shown in SEQ ID NO.17; 2 primers for amplifying RA have nucleotide sequences shown in SEQ ID NO.11 - 12; 1 probe for detecting RA has a nucleotide sequence shown in SEQ ID NO.

18.

2. The fluorescence PCR primer-probe set for simultaneously detecting GAstV and RA according to claim 1, wherein The 5′ end of the sequence of the probe for detecting GAstV is labeled with a fluorescent group FAM, and the 3′ end is labeled with a quenching group BHQ1.

3. The fluorescence PCR primer-probe set for simultaneously detecting GAstV and RA according to claim 1, characterized in that, The 5′ end of the sequence of the probe for detecting RA is labeled with a fluorescent group HEX, and the 3′ end is labeled with a quenching group BHQ1.

4. A kit containing the fluorescence PCR primer-probe set for simultaneously detecting GAstV and RA according to any one of claims 1 to 3, characterized in that, It includes the following reagents: (1) PCR reaction solution; (2) DNA polymerase, reverse transcriptase, dNTP mixture; (3) Primer and probe premixed solution: a mixed solution of the primers shown in SEQ ID NO.3 - 4, SEQ ID NO.11 - 12 and the probes shown in SEQ ID NO.17 - 18.

5. The kit according to claim 4, wherein It also contains the following reagents: (4) Positive control: an equal - volume mixed solution of the recombinant plasmid pMD - 3′UTR containing the GAstV gene and the recombinant plasmid pMD - pyrC containing the RA gene; (5) Negative control: purified water.

6. The kit according to claim 4, characterized in that, In the primer and probe premixed solution, the molar ratios of the primers shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.11 and SEQ ID NO.12 are 8:12:7:11, and the molar ratios of the probes shown in SEQ ID NO.17 and SEQ ID NO.18 are 5:

4.

7. The kit according to claim 4, characterized in that, The prepared concentrations of the primers shown in SEQ ID NO.3 - 4 and SEQ ID NO.11 - 12 are both 15 μmol / L, and the prepared concentrations of the probes shown in SEQ ID NO.17 - 18 are both 10 μmol / L.

8. The kit according to claim 5, wherein In the positive control, the concentration of plasmid pMD-3′UTR was 2.8×10 3 copies / μL, and the concentration of RA plasmid pMD-pyrC was 4.3×10 3 copies / μL.

9. The method of using the kit according to claim 4, wherein the method is for non-diagnostic and non-therapeutic purposes, and is characterized in that, It includes the following steps: Mix the sample to be tested with the PCR reaction solution, DNA polymerase, reverse transcriptase, dNTP mixture, and primer and probe premixed solution in the kit, and perform fluorescence PCR reaction according to the following reaction program: Reverse transcription at 42°C for 5 min; Pre - denaturation at 95°C for 10 s, denaturation at 95°C for 5 s, annealing at 55°C for 20 s, for a total of 40 cycles.

Images