Multiplex fluorescent quantitative PCR (polymerase chain reaction) detection composition, method and kit for detecting multiple RNA (ribonucleic acid) viruses in insect cells

Abstract

The invention discloses a multiplex fluorescent quantitative PCR (polymerase chain reaction) detection composition, method and kit for detecting multiple RNA (ribonucleic acid) viruses in insect cells, and the detection method is non-diagnostic or therapeutic and comprises the following steps: (1) extracting an RNA template of a sample to be detected; (2) establishing a fluorescent quantitative PCR reaction system by adopting the detection composition in claim 1; (3) carrying out fluorescent quantitative PCR (Polymerase Chain Reaction) detection; and (4) analyzing the result of the sample to be detected. According to the invention, a plurality of pairs of probe primers are designed for a virus conserved region, and the primer probes with good specificity and high sensitivity are finally screened, so that the virus content of 2 * 10 < 7 > copies / [mu] L to 20 copies / [mu] L can be accurately quantified. By repeatedly screening and optimizing a reaction system, including the concentrations of the primers and the probes, the detection method can stably detect various RNA viruses under the condition of fewer nucleic acid samples, is simple to operate and has higher sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biological detection, and in particular relates to a multiplex fluorescent quantitative PCR detection composition, method and kit for detecting multiple RNA viruses in insect cells. Background Art

[0002] Insect viruses, a class of viruses that use insects as hosts and can cause disease in insects, use insects as vectors, and can also infect humans and other mammals, causing epidemics and, in severe cases, death. Insect cells, including High Five cells, Sf9 cells, and D.mel-2 cells, are widely used in the biopharmaceutical field in the production of viral vaccines, recombinant proteins, and viral vectors. Therefore, exogenous viral infection in insect cells is considered a major safety concern.

[0003] Common RNA viruses that can infect insect cells include the Rhabdoviridae, Nodaviridae, Flaviviridae, and Bunyaviridae families, while more representative viruses include the Sf rhabdovirus reported to be able to infect the Sf cell line, a variant of the animal shed virus that can infect High Five cells, mammalian viruses including bovine viral diarrhea virus, classical swine fever virus, and Japanese encephalitis virus, as well as encephalomyocarditis virus, pine caterpillar ω virus, and whitefly fever virus.

[0004] Traditional methods for detecting insect viruses currently include biological assays, electron microscopy, enzyme-linked immunosorbent assay-based serological methods, and nucleic acid-based PCR. These methods generally suffer from low sensitivity and poor specificity. Furthermore, traditional qPCR methods often utilize separate detection kits, which are cumbersome, time-consuming, prone to false positives, or lack comprehensive coverage of viral species. Different virus detection reaction systems and procedures vary significantly, making them inadequate for simultaneous detection of multiple viruses. Furthermore, common RNA virus detection methods lack effective quality control materials during RNA extraction and reverse transcriptase. Traditional qPCR standards are often plasmids, which do not accurately reflect the status of RNA viruses. Summary of the Invention

[0005] The purpose of the present invention is to provide a multiplex fluorescent quantitative PCR detection composition, method and kit for detecting multiple RNA viruses in insect cells, which can accurately and quickly detect insect viruses in samples.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a multiplex fluorescent quantitative PCR detection composition for detecting multiple RNA viruses in insect cells, wherein the detection composition comprises primers and probes for detecting the following RNA viruses:

[0008] Primers and probe for detecting Sf rhabdovirus: the upstream primer is shown in SEQ ID NO: 1, the downstream primer is shown in SEQ ID NO: 2, and the probe is shown in SEQ ID NO: 3;

[0009] Primers and probe for detecting animal shed virus: the upstream primer is shown in SEQ ID NO: 4, the downstream primer is shown in SEQ ID NO: 5, and the probe is shown in SEQ ID NO: 6;

[0010] Primers and probe for detecting bovine viral diarrhea virus: the upstream primer is shown in SEQ ID NO: 7, the downstream primer is shown in SEQ ID NO: 8, and the probe is shown in SEQ ID NO: 9;

[0011] Primers and probes for detecting classical swine fever virus: the upstream primer is shown in SEQ ID NO: 10, the downstream primer is shown in SEQ ID NO: 11, and the probe is shown in SEQ ID NO: 12;

[0012] Primers and probe for detecting Japanese encephalitis virus: the upstream primer is shown in SEQ ID NO: 13, the downstream primer is shown in SEQ ID NO: 14, and the probe is shown in SEQ ID NO: 15;

[0013] Primers and probe for detecting Drosophila virus X: the upstream primer is shown in SEQ ID NO: 16, the downstream primer is shown in SEQ ID NO: 17, and the probe is shown in SEQ ID NO: 18;

[0014] Primers and probe for detecting encephalomyocarditis virus: the upstream primer is shown in SEQ ID NO: 19, the downstream primer is shown in SEQ ID NO: 20, and the probe is shown in SEQ ID NO: 21;

[0015] Primers and probe for detecting pine caterpillar ω virus: the upstream primer is shown in SEQ ID NO: 22, the downstream primer is shown in SEQ ID NO: 23, and the probe is shown in SEQ ID NO: 24;

[0016] Primers and probes for detecting sandfly fever virus: the upstream primer is shown in SEQ ID NO: 25, the downstream primer is shown in SEQ ID NO: 26, and the probe is shown in SEQ ID NO: 27.

[0017] In a second aspect, the present invention provides a multiplex fluorescent quantitative PCR detection kit for detecting multiple RNA viruses in insect cells, wherein the detection kit comprises the above-mentioned detection composition.

[0018] The fluorescent group of the probe is FAM, VIC or Cy5, and the quencher group is BHQ1 or BHQ2.

[0019] In the above technical solution, the detection kit includes a positive control substance, a negative control substance and a fluorescent quantitative PCR reaction reagent.

[0020] In the above technical solution, the fluorescent quantitative PCR reaction reagent is a fluorescent quantitative PCR reaction premix, including 2×One Step RT-PCR Buffer III, Ex Taq HS, RT Enzyme Mix II, ROX Reference Dye internal reference dye, nuclease-free water, and the above-mentioned detection composition.

[0021] In the above technical solution, the positive control contains positive RNA standards, and the concentration is 2×10 8 copies / μL.

[0022] In the above technical solution, the fluorescent quantitative PCR reaction system in the detection kit is 20 μL, including 15 μL of the fluorescent quantitative PCR reaction premix and 5 μL of template; the template is the RNA of the sample to be tested, the positive control standard and the negative control standard.

[0023] In the above technical solution, the reaction conditions of the reaction system are: inversion at 42°C for 5 min; pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 s, annealing at 60°C for 34 s, and 40 cycles.

[0024] In a third aspect, the present invention provides a multiplex fluorescence quantitative PCR detection method for detecting multiple RNA viruses in insect cells, wherein the detection method is not for diagnostic or therapeutic purposes and comprises the following steps:

[0025] (1) extracting RNA template from the sample to be tested;

[0026] (2) establishing a fluorescent quantitative PCR reaction system using the above-mentioned detection composition;

[0027] (3) Perform fluorescence quantitative PCR detection;

[0028] (4) Analysis of test results of the samples.

[0029] In the above technical solution, the fluorescent quantitative PCR reaction system is 20 μL, including 15 μL of fluorescent quantitative PCR reaction premix and 5 μL of template; the template is the RNA of the sample to be tested, a positive control standard and a negative control standard; the fluorescent quantitative PCR reaction premix includes 2×One Step RT-PCR Buffer III, Ex Taq HS, RT Enzyme Mix II, ROX Reference Dye internal reference dye, nuclease-free water, and the above-mentioned detection composition.

[0030] In the above technical solution, the reaction conditions of the fluorescent quantitative PCR reaction system are: inversion at 42°C for 5 min; pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 s, annealing at 60°C for 34 s, and 40 cycles.

[0031] The beneficial effects of the present invention are:

[0032] 1. The present invention aims to detect a variety of RNA viruses that can infect insect cells. By analyzing and comparing databases, we can find conserved sequences in the viral genome and design primer probes with strong specificity to detect most subtypes of the virus.

[0033] 2. The detection composition of the present invention, wherein the probe fluorescent reporter group can be selected from FAM, VIC or Cy5, and the fluorescent quencher group can be selected from BHQ1 or BHQ2, can meet the requirements of detecting any one of the above-mentioned RNA viruses, and can also detect any combination of RNA viruses.

[0034] 3. By designing multiple pairs of probe primers for the conserved regions of the virus, we can finally screen the primer probes with good specificity and high sensitivity, which can accurately quantify 2×10 7 The virus content ranges from copies / μL to 20 copies / μL. The detection sensitivity of most viruses can reach 10 copies / μL, and the minimum sensitivity of some viruses can reach 2 copies / μL.

[0035] 4. The detection kit for detecting multiple RNA viruses in insect cells of the present invention contains positive RNA standards for detection, which can more realistically reflect the RNA virus detection process and test the RNA qPCR detection method at the same time, and can meet the requirements of simultaneous detection of multiple RNA viruses in one program.

[0036] 5. The present invention repeatedly screens and optimizes the reaction system, including the concentrations of primers and probes. Ultimately, the detection method can stably detect multiple RNA viruses with relatively small nucleic acid samples. It is simple to operate and has high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1qPCR amplification curve for SFRV detection;

[0038] Figure 2 qPCR standard curve for SFRV detection;

[0039] Figure 3 qPCR amplification curve for FHV detection;

[0040] Figure 4 qPCR standard curve for FHV detection;

[0041] Figure 5 qPCR amplification curve for BVDV detection;

[0042] Figure 6 qPCR standard curve for BVDV detection;

[0043] Figure 7 qPCR amplification curve for CSFV detection;

[0044] Figure 8 qPCR standard curve for CSFV detection;

[0045] Figure 9 qPCR amplification curve for JEV detection;

[0046] Figure 10 qPCR standard curve for JEV detection;

[0047] Figure 11 qPCR amplification curve for DXV detection;

[0048] Figure 12 qPCR standard curve for DXV detection;

[0049] Figure 13 qPCR amplification curve for EMCV detection;

[0050] Figure 14 qPCR standard curve for EMCV detection;

[0051] Figure 15 qPCR amplification curve for NωV detection;

[0052] Figure 16 qPCR standard curve for NωV detection;

[0053] Figure 17 This is the qPCR amplification curve for SFV detection;

[0054] Figure 18 qPCR standard curve for SFV detection. DETAILED DESCRIPTION

[0055] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the present invention to those skilled in the art. The present invention will be limited only by the claims.

[0056] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0057] The present invention will now be further described in conjunction with specific examples. The following examples are only intended to explain the present invention but do not limit the present invention. The test samples and test procedures used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually based on conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources).

[0058] The experimental materials used in the examples of the present invention are:

[0059] Premix and ROX were manufactured by TAKARA, and nuclease-free water was manufactured by Invitrogen;

[0060] RNA extraction kit: purchased from Tiangen, product number: DP419;

[0061] One-Step PrimeScript RT-PCR Kit (containing ROX Reference Dye) was purchased from TaKaRa, catalog number: RR064A.

[0062] Nuclease-free water was purchased from Invitrogen, catalog number: 10977-015.

[0063] Example 1 Primer probe design and synthesis

[0064] By consulting relevant standards, references, and the NCBI database, we confirmed relevant viral genome sequence information, selected representative viral genome sequences, and selected conserved genomic regions to design and test highly specific and sensitive primer and probe sequences. Information on relevant RNA viruses is shown in Table 1.

[0065] Table 1 RNA virus information

[0066]

[0067]

[0068] The primer and probe sequences for fluorescence quantitative PCR detection are shown in Table 2.

[0069] Table 2 Primer and probe sequence information

[0070]

[0071]

[0072] At the same time, a representative viral genome sequence was selected, and sequence fragments were selected for the conserved viral regions designed for primer probes. The fragments were constructed into the pRNA-SP6-T7 plasmid vector to obtain the corresponding plasmid. RNA was transcribed using the T7 promoter to obtain the corresponding RNA standard.

[0073] The RNA sequences are shown in Table 3.

[0074] Table 3 Synthetic RNA sequence information

[0075]

[0076]

[0077]

[0078]

[0079] Example 2 Multiplex qPCR Detection Method

[0080] Positive RNA standards were used to screen the primers and the RNA standards were diluted 10-fold in a series of 7 dilutions, with a dilution of 2×10 7 copies / μL(STD01), 2×10 6 copies / μL(STD02), 2×10 5 copies / μL(STD03), 2×10 4 copies / μL(STD04), 2×10 3 copies / μL(STD05), 2×10 2 copies / μL (STD06) and 2×10 1There are 7 different concentrations of standard samples, 10 copies / μL (STD07), and 5 μL of nuclease-free water is used as a negative control template. The qPCR reaction premix configuration is shown in Table 4 below:

[0081] Table 4 qPCR reaction system

[0082] Components Volume (μl) 2XOneStepRT-PCRBufferIII 10.0 TaKaRaExTaqHS (5U / μL) 0.4 PrimeScript RT Enzyme Mix II 0.4 Forward primer (10 μM) 0.4 Reverse primer (10 μM) 0.4 Probe (10 μM) 0.2 50×ROX 0.4 Nuclease-free water 2.8 Standards / test samples 5.0

[0083] Use an ABI 7500 PCR instrument and set up the qPCR reaction program according to Table 5, collecting signals at 60°C:

[0084] Table 5 qPCR reaction procedure

[0085]

[0086]

[0087] Example 3 A Multiplex Fluorescence Quantitative PCR Detection Kit for Detecting Multiple RNA Viruses in Insect Cells

[0088] Based on the above detection method, the results of this method are shown in Tables 6 to 14. The standard curves obtained by the primer probes all meet the acceptance criteria (R 2 >0.99, 90%≤Eff%≤110%, recovery rate between 50%-150%). The results are analyzed in Tables 6-14.

[0089] Table 6 Analysis of SFRV calibration results

[0090]

[0091] UD: undetermined; NA: Not applicable

[0092] Table 7 Analysis of FHV calibration results

[0093]

[0094]

[0095] UD: undetermined; NA: Not applicable

[0096] Table 8 Analysis of BVDV calibration results

[0097]

[0098]

[0099] UD: undetermined; NA: Not applicable

[0100] Table 9 CSFV standard curve results analysis

[0101]

[0102] UD: undetermined; NA: Not applicable

[0103] Table 10 Analysis of JEV calibration curve results

[0104]

[0105] UD: undetermined; NA: Not applicable

[0106] Table 11 DXV marking result analysis

[0107]

[0108]

[0109] UD: undetermined; NA: Not applicable

[0110] Table 12 EMCV calibration curve results analysis

[0111]

[0112]

[0113] UD: undetermined; NA: Not applicable

[0114] Table 13 Analysis of NωV calibration results

[0115]

[0116] UD: undetermined; NA: Not applicable

[0117] Table 14 SFV curve analysis results

[0118]

[0119]

[0120] UD: undetermined; NA: Not applicable

[0121] According to Table 6-Table 14 and Figures 1-18 It can be seen that the RNA template copy number is 108 ~10 2 When the range is within 200 nm, the established fluorescence quantitative PCR detection method has a good amplification curve, and the correlation coefficient R 2 =0.999, the amplification efficiency is 97.078%; the correlation coefficient of FHV R 2 =0.999, the amplification efficiency is 97.078%; the correlation coefficient of BVDV is R 2 =1.000, the amplification efficiency is 92.881%; the correlation coefficient R 2 =1.000, the amplification efficiency is 95.910%; the correlation coefficient of JEV is R 2 =0.999, the amplification efficiency is 92.132%; the correlation coefficient of DXV is R 2 =0.999, the amplification efficiency is 97.192%; the correlation coefficient of EMCV is R 2 =0.999, the amplification efficiency is 99.565%; the correlation coefficient of NωV is R 2 =1.000, the amplification efficiency is 93.150%; the correlation coefficient of SFV is R 2 =1.000, and the amplification efficiency is 92.532%.

[0122] Example 4 Kit Detection Limit Test

[0123] The detection limit refers to the lowest number of virus copies that can be detected in a sample. Based on the previous primer probe screening and test results, the detection limit of the above viruses was tested on 10 3 copies, 10 2 The groups of 100 copies, 50 copies and 10 copies / reaction were set, and 6 replicates were set for each group to verify the minimum detection limit of the method. The detection limit results are shown in Table 15.

[0124] Table 15 Analysis of minimum detection limit results

[0125]

[0126]

[0127] The minimum detection limits (MLDs) are shown in Table 15. The MLD for this assay requires detection in all six replicates. The MLDs for BVDV were 100 copies / reaction, SFRV, FHV, and DXV were 50 copies / reaction, and CSFV, JEV, EMCV, NωV, and SFV were 10 copies / reaction.

[0128] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A multiplex fluorescence quantitative PCR detection composition for detecting multiple RNA viruses in insect cells, characterized in that: The detection composition includes primers and probes for detecting the following RNA viruses: Primers and probe for detecting Sf rhabdovirus: the upstream primer is shown in SEQ ID NO: 1, the downstream primer is shown in SEQ ID NO: 2, and the probe is shown in SEQ ID NO: 3; Primers and probes for detecting animal shed virus: the upstream primer is shown in SEQ ID NO: 4, the downstream primer is shown in SEQ ID NO: 5, and the probe is shown in SEQ ID NO: 6; Primers and probes for detecting bovine viral diarrhea virus: the upstream primer is shown in SEQ ID NO: 7, the downstream primer is shown in SEQ ID NO: 8, and the probe is shown in SEQ ID NO: 9; Primers and probes for detecting classical swine fever virus: the upstream primer is shown in SEQ ID NO: 10, the downstream primer is shown in SEQ ID NO: 11, and the probe is shown in SEQ ID NO: 12; Primers and probes for detecting Japanese encephalitis virus: the upstream primer is shown in SEQ ID NO: 13, the downstream primer is shown in SEQ ID NO: 14, and the probe is shown in SEQ ID NO: 15; Primers and probe for detecting Drosophila X virus: the upstream primer is shown in SEQ ID NO: 16, the downstream primer is shown in SEQ ID NO: 17, and the probe is shown in SEQ ID NO: 18; Primers and probes for detecting encephalomyocarditis virus: the upstream primer is shown in SEQ ID NO: 19, the downstream primer is shown in SEQ ID NO: 20, and the probe is shown in SEQ ID NO: 21; Primers and probes for detecting pine moth ω virus: the upstream primer is shown in SEQ ID NO: 22, the downstream primer is shown in SEQ ID NO: 23, and the probe is shown in SEQ ID NO: 24; Primers and probes for detecting sandfly fever virus: the upstream primer is shown in SEQ ID NO: 25, the downstream primer is shown in SEQ ID NO: 26, and the probe is shown in SEQ ID NO:

27.

2. A multiplex fluorescence quantitative PCR detection kit for detecting multiple RNA viruses in insect cells, characterized in that: The detection kit comprises the detection composition according to claim 1.

3. The detection kit according to claim 2, characterized in that: The detection kit comprises a positive control substance, a negative control substance and a fluorescent quantitative PCR reaction reagent.

4. The detection kit according to claim 3, characterized in that: The fluorescent quantitative PCR reaction reagent is a fluorescent quantitative PCR reaction premix, which includes 2×One Step RT-PCR Buffer III, Ex Taq HS, RT Enzyme Mix II, ROX Reference Dye internal reference dye, nuclease-free water, and the detection composition according to claim 1.

5. The detection kit according to claim 3, characterized in that: The positive control contains positive RNA standards, all with a concentration of 2×10 8 copies / μL.

6. The detection kit according to claim 4, characterized in that: The fluorescence quantitative PCR reaction system in the detection kit is 20 μL, including 15 μL of the fluorescence quantitative PCR reaction premix and 5 μL of template; the template is the RNA of the sample to be tested, the positive control standard and the negative control standard.

7. The detection kit according to claim 6, characterized in that: The reaction conditions of the reaction system are: inversion at 42° C. for 5 min; pre-denaturation at 95° C. for 3 min; denaturation at 95° C. for 5 s, annealing at 60° C. for 34 s, and 40 cycles.

8. A multiplex fluorescence quantitative PCR detection method for detecting multiple RNA viruses in insect cells, characterized in that: The detection method is not for diagnosis or treatment purposes and comprises the following steps: (1) extracting RNA template of the sample to be tested; (2) using the detection composition of claim 1 to establish a fluorescent quantitative PCR reaction system; (3) Conducting fluorescence quantitative PCR detection; (4) Analysis of test results of samples.

9. The detection method according to claim 8, characterized in that: The fluorescent quantitative PCR reaction system is 20 μL, including 15 μL of fluorescent quantitative PCR reaction premix and 5 μL of template; the template is RNA of the sample to be tested, a positive control standard and a negative control standard; the fluorescent quantitative PCR reaction premix includes 2×One Step RT-PCR BufferⅢ, ExTaq HS, RT Enzyme MixⅡ, ROX Reference Dye internal reference dye, nuclease-free water, and the detection composition according to claim 1.

10. The detection method according to claim 8, characterized in that: The reaction conditions of the fluorescent quantitative PCR reaction system are: inversion at 42° C. for 5 min; pre-denaturation at 95° C. for 3 min; denaturation at 95° C. for 5 s, annealing at 60° C. for 34 s, and 40 cycles.

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