Fluorescent quantitative PCR (Polymerase Chain Reaction) primer group for detecting mandarin fish arenavirus and application

Abstract

The invention relates to a fluorescent quantitative PCR primer group for detecting mandarin fish arenavirus and application, and relates to the technical field of biological detection. The fluorescent quantitative PCR primer group comprises an amplification primer pair as shown in SEQ ID NO. 1 and SEQ ID NO. 2, and a probe as shown in SEQ ID NO. 3. On the basis of the fluorescent quantitative PCR primer group, the mandarin fish arenavirus MFAV can be well amplified and detected, the detection result has good specificity and sensitivity, cross reaction with other viruses capable of infecting mandarin fish except the mandarin fish arenavirus is avoided, and the lowest detection concentration is 2.20 * 10 < 2 > copies / mu L.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, in particular to a fluorescence quantitative PCR primer set for detecting Mandarin fish arenavirus and its application. Background Art

[0002] Mandarin fish arenavirus (MFAV) is a new type of aquatic virus discovered in recent years. We detected MFAV in Mandarin fish in Guangdong Province. At present, there is little research on MFAV, and there are no related detection methods or diagnostic kits. Therefore, it is crucial to develop a technology and kit that can accurately, quickly, and effectively identify MFAV for the prevention and control of this virus. Summary of the Invention

[0003] In view of the above problems, the present invention provides a fluorescence quantitative PCR primer set for detecting Mandarin fish arenavirus. Based on this fluorescence quantitative PCR primer set, good amplification and detection of MFAV can be achieved. The detection results have good specificity and sensitivity, and there is no cross-reaction with other viruses that can infect Mandarin fish except MFAV. The lowest detection concentration is 2.20×10 2 copies / μL.

[0004] The present invention provides a fluorescence quantitative PCR primer set for detecting Mandarin fish arenavirus, including an amplification primer pair and a probe; the amplification primer set is as follows:

[0005] MFAV-F: 5’-GGATTCTTTACTGTGCGCCA-3’ (SEQ ID NO.1);

[0006] MFAV-R: 5’-TTGCACTCCAAACTGACCAC-3’ (SEQ ID NO.2);

[0007] The probe is as follows:

[0008] MFAV-probe: 5’-AATGGGACCCTGAGCTACCTCACTG-3’ (SEQ ID NO.3).

[0009] In one embodiment, the 5’ end of the probe is connected to a fluorescent reporter group FAM, and the 3’ end of the probe is connected to a fluorescent quenching group BHQ1.

[0010] The present invention also provides a detection system for detecting Mandarin fish arenavirus, including a fluorescence quantitative PCR detection reaction system, and the fluorescence quantitative PCR detection reaction system includes the above-mentioned fluorescence quantitative PCR primer set.

[0011] In one embodiment, the detection system further includes ROX reference dye.

[0012] The present invention also provides a kit for detecting Siniperca chuatsi sand grain virus, which includes the above-mentioned fluorescence quantitative PCR primer set, or includes the above-mentioned detection system.

[0013] The amplification curve of the above-mentioned kit is good, and it has the advantages of simple operation method, strong specificity, high sensitivity, and short time consumption of the reaction results.

[0014] In one embodiment, the kit further includes a positive control and a negative control. The positive control includes the glycoprotein gene of Siniperca chuatsi sand grain virus.

[0015] In one embodiment, the negative control is DEPC-treated ddH2O.

[0016] The present invention also provides a method for detecting Siniperca chuatsi sand grain virus, which includes the following steps: extracting the RNA of the sample to be detected, reverse transcribing it into cDNA, and performing fluorescence quantitative PCR amplification using the above-mentioned fluorescence quantitative PCR primer set, the above-mentioned detection system or the above-mentioned kit.

[0017] In one embodiment, the reaction conditions of the fluorescence quantitative PCR amplification include: 95°C for 3 min; 95°C for 2 s; 60°C for 20 s; 45 cycles; collecting fluorescence signals during the extension at 60°C.

[0018] The present invention also provides the application of the primer set, the detection system or the kit in the preparation of products for detecting Siniperca chuatsi sand grain virus.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The fluorescence quantitative PCR primer set and application for detecting Siniperca chuatsi sand grain virus of the present invention can achieve better amplification and detection of Siniperca chuatsi sand grain virus MFAV based on this fluorescence quantitative PCR primer set. The detection results have good specificity and sensitivity, and there is no cross-reaction with other viruses that can infect Siniperca chuatsi except Siniperca chuatsi sand grain virus. The lowest detection concentration is 2.20×10 2 copies / μL. Description of the Drawings

[0021] Figure 1 For the establishment of the amplification curve of the TaqMan fluorescence quantitative PCR detection method for Siniperca chuatsi MFAV, where ① is the amplification curve of the MFAV genome and ② is the amplification curve of the negative control;

[0022] Figure 2For the specific detection of the MFAV TaqMan fluorescence quantitative PCR detection method for mandarin fish, in the figure: ① represents the amplification curve of the MFAV genome; ② represents the amplification curves of other viruses that can infect mandarin fish (including the genomes of nervous necrosis virus (RGNNV) and grouper iridovirus (SGIV), and negative control).

[0023] Figure 3 When using TaqMan fluorescence quantitative PCR to detect the sensitivity of MFAV virus, the RNA concentrations of MFAV virus are 2.20×10 6 copies / μL to 2.20×10 2 copies / μL 10-fold serial dilution positive standard products and the corresponding amplification curves of negative control;

[0024] Figure 4 It is the standard curve drawn by gradient dilution of positive standard products. Specific implementation mode

[0025] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0027] Source:

[0028] Unless otherwise specified, the reagents, materials, and equipment used in this embodiment are all commercially available; unless otherwise specified, the experimental methods are all conventional experimental methods in this field.

[0029] Example 1

[0030] 1. Design specific primers and probes for the conserved region of the glycoprotein gene of MFAV.

[0031] Download the reference sequence of the MFAV strain from GenBank. According to the sequence alignment results, design a pair of specific primers (MFAV-F / MFAV-R, the length of the amplified target fragment is 915 bp) and the corresponding TaqMan probe (MFAV-probe) for the highly conserved fragment of the glycoprotein gene of MFAV:

[0032] MFAV-F: 5’-GGATTCTTTACTGTGCGCCA-3’ (SEQ ID NO.1);

[0033] MFAV-R: 5’-TTGCACTCCAAACTGACCAC-3’ (SEQ ID NO.2);

[0034] MFAV-probe: 5’-AATGGGACCCTGAGCTACCTCACTG-3’ (SEQ ID NO.3). Label the 5’ end of the probe with the fluorescent reporter group FAM and the 3’ end with the fluorescent quenching group BHQ1.

[0035] 2. Establishment of the TaqMan fluorescence quantitative PCR detection method for Siniperca chuatsi MFAV.

[0036] According to the instructions of the viral DNA / RNA small-scale preparation kit, extract MFAV RNA and reverse transcribe it into cDNA as the template, and use the following primer concentration, probe concentration and reaction conditions. The reaction system is shown in Table 1.

[0037] Table 1 TaqMan fluorescence quantitative PCR detection system for MFAV

[0038]

[0039] Reaction conditions: 95°C (pre-denaturation) for 3 min; 95°C (denaturation) for 2 s, 60°C (annealing) for 20 s, 45 cycles (PCR amplification); collect fluorescence signals during the extension at 60°C, and set the reporter gene as FAM.

[0040] 3. Fluorescence quantitative detection.

[0041] In the fluorescence quantitative detection, add various components in Table 1 to the detection system of the glycoprotein gene of MFAV. After mixing all components evenly, enter the cycle, and detect the fluorescence signal for 45 cycles. Use the fluorescence detection to determine the detection activity of the detection system. Using the fluorescence method result determination scheme, the detection of MFAV can be achieved.

[0042] The fluorescence quantitative PCR detection system in Table 1 and the corresponding reaction conditions were used to detect the MFAV virus. Using the cDNA of the MFAV genome as a template, the above primers were used for PCR amplification of the target fragment. As Figure 1 shown, a relatively high fluorescence value was found in the detection of the MFAV virus.

[0043] Based on the above results, this system and method had a relatively high fluorescence value for the detected MFAV virus genome. Therefore, this system was used for subsequent detection. This detection used a 20 μL system as shown in Table 1, but was not limited thereto.

[0044] Example 2

[0045] Specific detection of the MFAV virus.

[0046] The samples contained: the genome of mandarin fish sand grain virus (MFAV), nervous necrosis virus (RGNNV), the genome of grouper iridovirus (SGIV), and negative control.

[0047] After separately extracting the genomic RNA of each virus and reverse transcribing it into cDNA, the established TaqMan fluorescence quantitative PCR detection method for mandarin fish MFAV in Example 1 was used for detection. Except for MFAV, no specific amplification signals were detected in other samples ( Figure 2 ), indicating that the detection method established in Example 1 had good specificity.

[0048] Example 3

[0049] Detection of the sensitivity of the MFAV virus.

[0050] In the sensitivity detection, the pcDNA3.1-MFAV-glycoprotein recombinant plasmid of MFAV (obtained by ligating the target fragment of the glycoprotein gene of mandarin fish sand grain virus to pcDNA3.1) was converted into copy numbers according to the molecular weight, reverse transcribed after 10-fold serial dilution, and the positive standard product was made at a concentration of 2.20×10 6 copies / μL to 2.20×10 2 copies / μL. The established TaqMan fluorescence quantitative PCR detection method for mandarin fish MFAV in Example 1 was used for detection. The sensitivity of the established method could reach 2.20×10 2 copies / μL ( Figure 3 ), and the sensitivity was good.

[0051] The results obtained in this example were as Figure 3 shown. For the sensitivity detection results of the MFAV virus, the sensitivity of the established method could reach 2.20×10 2copies / μL, with good sensitivity, and can be applied to primary detection.

[0052] Example 4

[0053] Based on the copy number logarithm values obtained in Example 3 and the quantitative detection ct values, a standard curve was plotted. As Figure 4 shown, the fitting curve equation is y = -3.5622x + 46.686, R 2 = 0.9838, with good fitting effect, and can be used for the quantitative detection of MFAV virus.

[0054] Example 5

[0055] Quantitative detection of the sample to be tested.

[0056] 1. Preparation of RNA template.

[0057] 1.1 Preparation of plasmid RNA template.

[0058] The positive recombinant plasmid pcDNA3.1-MFAV-glycoprotein of MFAV was synthesized and constructed by Wuhan Genecreate Biological Engineering Co., Ltd., which was obtained by ligating the target fragment of the glycoprotein gene of Siniperca chuatsi rhabdovirus to pcDNA3.1. The plasmid contains the T7 promoter region. The RNA transcript of MFAV glycoprotein was prepared according to the T7 in vitro transcription kit. The specific reagent components and dosages are shown in Table 2. Incubate at 37 °C for 2 h. After complete incubation, add 2 μL of RNase free DNase I and digest at 37 °C for 30 min to remove residual DNA.

[0059] Table 2 In vitro transcription reaction system

[0060]

[0061]

[0062] 1.2 Preparation of RNA template of the sample to be tested.

[0063] Using FastPure Viral DNA / RNA Mini Kit (Vazyme), extract the genomic RNA of the sample. The specific method is as follows:

[0064] (1) Add 200 μL of the sample to an RNase-free centrifuge tube (if the sample volume is insufficient, make up with PBS or 0.9% NaCl). Add 500 μL of Buffer VL, vortex for 15 - 30 s, and centrifuge the mixture instantaneously to collect it at the bottom of the tube.

[0065] (2) Place the FastPure RNA Columns in a 2 mL collection tube, transfer the above mixture to the FastPure RNA Columns, centrifuge at 12,000×g for 1 min, and discard the filtrate.

[0066] (3) Add 600 μL of Buffer RW to the FastPure RNA Columns, centrifuge at 12,000×g for 30 s, and discard the filtrate. Repeat step 3. Centrifuge the empty column at 12,000×g for 2 min.

[0067] (4) Carefully transfer the FastPure RNA Columns to a new 1.5 mL RNase-free collection tube (provided in the kit), add 30 - 50 μL of RNase-free dH2O to the center of the membrane in a suspended manner, let it stand at room temperature for 1 min, and centrifuge at 12,000×g for 1 min.

[0068] (5) Discard the FastPure RNA Columns. The RNA can be directly used for subsequent detection or stored at -30 to -15 °C for short-term or at -85 °C for long-term.

[0069] 2. Preparation of cDNA template Reverse transcribe the above-prepared RNA template into a cDNA template, and the reaction system is shown in Table 3. The reaction program is: react at 30 °C for 10 min, react at 42 °C for 40 min, then inactivate at 95 °C for 5 min, and store the product at -20 °C for standby.

[0070] Table 3 Reverse transcription reaction system

[0071]

[0072] 3. Make serial dilutions of the positive standard (positive recombinant plasmid pcDNA3.1-MFAV-glycoprotein of MFAV) from 2.20×10 6 copies / μL to 2.20×10 2 copies / μL, and then prepare cDNA as a template according to the methods in 1.1 and 2 above, and perform fluorescence quantitative PCR amplification with the specific primers, probes, reaction system, and reaction conditions described in Example 1 to establish a standard curve.

[0073] 4. Prepare the cDNA template for the sample to be tested according to the methods in 1.2 and 2 above, and perform fluorescence quantitative PCR amplification with the specific primers, probes, reaction system, and reaction conditions described in Example 1.

[0074] 5. Quantify the viral copy number in the sample to be tested according to the standard curve ( Figure 4 ) If it is for qualitative detection, there is no need to construct a standard curve.

[0075] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0076] The above-described embodiments only express several implementation manners of the present invention. The description is relatively specific and detailed, but it cannot be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A fluorescent quantitative PCR primer set for detecting mandarin fish arenavirus, characterized in that: It includes an amplification primer pair and a probe; the amplification primer set is as follows: MFAV-F: 5'-GGATTCTTTACTGTGCGCCA-3' (SEQ ID NO. 1); MFAV-R: 5'-TTGCACTCCAAACTGACCAC-3' (SEQ ID NO. 2); The probe is as follows: MFAV-probe: 5'-AATGGGACCCTGAGCTACCTCACTG-3' (SEQ ID NO. 3).

2. The fluorescent quantitative PCR primer set according to claim 1, characterized in that: The 5' end of the probe is connected to a fluorescent reporter group FAM, and the 3' end of the probe is connected to a fluorescent quencher group BHQ1.

3. A detection system for detecting mandarin fish arenavirus, characterized in that: It comprises a fluorescence quantitative PCR detection reaction system, wherein the fluorescence quantitative PCR detection reaction system comprises the fluorescence quantitative PCR primer set according to any one of claims 1-2.

4. The detection system according to claim 3, characterized in that: The detection system also includes a ROX reference dye.

5. A kit for detecting mandarin fish arenavirus, characterized in that: The invention comprises the fluorescent quantitative PCR primer set according to any one of claims 1 to 2, or comprises the detection system according to any one of claims 3 to 4.

6. The kit according to claim 5, characterized in that The kit also includes a positive control substance and a negative control substance, wherein the positive control substance includes the glycoprotein gene of the mandarin fish arenavirus.

7. A method for detecting mandarin fish arenavirus for non-disease diagnosis purposes, characterized in that: The following steps are involved: The RNA of the sample to be tested is extracted and reverse transcribed into cDNA, and fluorescent quantitative PCR amplification is performed using the fluorescent quantitative PCR primer set described in any one of claims 1-2, the detection system described in any one of claims 3-4, or the kit described in any one of claims 5-6.

8. The detection method according to claim 7, characterized in that: The reaction conditions of the fluorescent quantitative PCR amplification include: 95° C., 3 min; 95° C., 2 s; 60° C., 20 s; 45 cycles; and collecting the fluorescent signal during the extension at 60° C.

9. Use of the primer set according to any one of claims 1 to 2, the detection system according to any one of claims 3 to 4, or the kit according to any one of claims 5 to 6 in the preparation of a product for detecting mandarin fish arenavirus.

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