A multiplex fluorescent quantitative PCR detection composition, method and kit for detecting multiple RNA viruses in insect cells

Abstract

The application discloses a multiplex fluorescent quantitative PCR detection composition, method and kit for detecting multiple RNA viruses in insect cells, and the detection method is not for diagnosis or treatment purposes, and comprises the following steps: (1) extracting RNA templates of samples to be detected; (2) establishing a fluorescent quantitative PCR reaction system by using the detection composition in claim 1; (3) performing fluorescent quantitative PCR detection; and (4) analyzing results of the samples to be detected. The application designs multiple pairs of probe primers for a conserved region of viruses, finally screens primers and probes with good specificity and high sensitivity, and can accurately quantify virus contents of 2*10 7 copies / µL to 20 copies / µL. The application repeatedly screens and optimizes a reaction system, including concentrations of primers and probes, finally the detection method can stably detect multiple RNA viruses under less nucleic acid samples, is simple to operate, and has high sensitivity.

Description

TECHNICAL FIELD

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

[0002] Insect viruses are a class of viruses that use insects as hosts and can cause diseases in insects. They can also infect humans and other mammals, causing epidemics and even death. Insect cells are widely used in the field of biological medicine, including High Five cells, Sf9 cells, D. mel-2 cells, which are often used for 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 Rhabdoviridae, Nodaviridae, Flaviviridae, and Bunyaviridae. More representative viruses include Sf rhabdovirus, which is reported to infect Sf cell lines, and the variant of the vesicle virus, which can infect High Five cells. Mammalian viruses, including bovine viral diarrhea virus, swine fever virus, and Japanese encephalitis virus, as well as encephalomyocarditis virus, pine caterpillar omega virus, and phlebotomus fever virus.

[0004] Current traditional insect virus detection methods generally include biological assays, electron microscopy observation, serological methods based on enzyme-linked immunosorbent assay, and PCR detection methods based on nucleic acid analysis. These methods generally have low detection sensitivity and poor specificity. Traditional qPCR methods often use separate detection kits, which are cumbersome to operate, time-consuming, and prone to false positives or failure to cover a wide range of virus species. Different virus detection reaction systems and procedures differ greatly, and cannot meet the needs of simultaneous detection of multiple viruses. Common RNA virus detection methods lack good quality control products during RNA extraction and reverse transcription. Traditional qPCR selects plasmids as standard products, which cannot accurately reflect the state of RNA viruses. SUMMARY

[0005] The present application provides 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] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a multiplex fluorescent quantitative PCR detection composition for detecting a plurality of RNA viruses in insect cells, the detection composition comprising primers and probes for detecting the following RNA viruses:

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

[0009] Primers and probes for detecting the stable fly virus: the upstream primer is shown as SEQ ID NO: 4, the downstream primer is shown as SEQ ID NO: 5, and the probe is shown as SEQ ID NO: 6;

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

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

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

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

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

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

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

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

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

[0019] In the above technical solution, the detection kit comprises a positive control, a negative control 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, comprising 2x One Step RT-PCR Buffer III, Ex Taq HS, RT Enzyme Mix II, ROX Reference Dye internal reference dye, RNase-free water and the above detection composition.

[0021] In the above technical solution, the positive control comprises a positive RNA standard, and the concentration of each is 2x10 8 copies / μL.

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

[0023] In the above technical solution, the reaction conditions of the reaction system are as follows: 42℃ reverse for 5 min; 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s, 60℃ annealing for 34 s, 40 cycles.

[0024] In a third aspect, the present application provides a multiplex fluorescent quantitative PCR detection method for detecting multiple RNA viruses in insect cells, which is not for diagnostic or therapeutic purposes, comprising the following steps:

[0025] (1) extracting an RNA template of a sample to be detected;

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

[0027] (3) performing fluorescent quantitative PCR detection;

[0028] (4) analyzing the results of the sample to be detected.

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

[0030] In the above technical scheme, the reaction conditions of the fluorescent quantitative PCR reaction system are: 42 DEG C reverse 5 min; 95 DEG C pre-denaturation 3 min; 95 DEG C denaturation 5 s, 60 DEG C annealing 34 s, 40 cycles.

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

[0032] 1. The present application aims to detect a plurality of RNA viruses capable of infecting insect cells, find conserved sequences of viral genomes through database analysis and comparison, design primers and probes with strong specificity, and detect most subtypes of the viruses.

[0033] 2. The detection composition of the present application, wherein the probe fluorescent reporter group can be selected from FAM, VIC or Cy5, and the fluorescent quenching group can be selected from BHQ1 or BHQ2, so as to meet the detection of any one of the above-mentioned RNA viruses, and simultaneously detect any combination of RNA viruses.

[0034] 3. By designing multiple pairs of probe primers in the conserved region of the virus, the primers and probes with good specificity and high sensitivity are finally screened, and the viral content of 2x10 7 copies / muL to 20 copies / muL can be accurately quantified, and the detection sensitivity of most viruses can reach 10 copies / muL, and the minimum sensitivity of some viruses can reach 2 copies / muL.

[0035] 4. The detection kit for detecting a plurality of RNA viruses in insect cells of the present application contains a positive RNA standard for detection, can more truly reflect the RNA virus detection process, simultaneously tests the qPCR detection method of RNA, and can meet the simultaneous detection of a plurality of RNA viruses in one program.

[0036] 5. The present application optimizes the reaction system including the concentration of primers and probes through repeated screening, and finally the detection method can stably detect a plurality of RNA viruses under less nucleic acid sample, is simple to operate, and has high sensitivity. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0053] Figure 17 qPCR amplification curve for detection of SFV;

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

[0055] For the purpose of better illustrating the object, technical solutions and advantages of the present application, the present application will be further described in the following with specific examples. The present application can be implemented in many different forms, and should not be understood as being limited to the examples set forth herein. On the contrary, these examples are provided so that the present disclosure will be thorough and complete, and will fully convey the inventive concept of the present application to those skilled in the art, which will be limited only by the claims.

[0056] It should be noted that the terms used herein are only intended to describe specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form, unless the context clearly indicates otherwise, and it should also be understood that, when the terms "comprise" and / or "include" are used in the specification, there is a presence of a feature, step, operation, device, component and / or combination thereof.

[0057] The present application will be further described with specific examples, which are only intended to explain the present application, but do not constitute a limitation on the present application. The test samples and test procedures used in the following examples include the following contents (if the specific conditions of the experiments are not specified in the examples, the conventional conditions or the conditions recommended by the reagent company are generally used; the reagents, consumables, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified).

[0058] Experimental materials used in the embodiments of the present application:

[0059] The premix and ROX manufacturer is TAKARA, and the nuclease-free water manufacturer is Invitrogen;

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

[0061] One Step PrimeScript RT-PCR Kit for probe method one-step qPCR special reagent purchased from TaKaRa (containing ROX Reference Dye internal standard dye), item number: RR064A;

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

[0063] Example 1 primer probe design and synthesis

[0064] By consulting relevant standards, reference literature, through NCBI database, confirming relevant viral genome sequence information, selecting representative viral genome sequence, selecting genomic conserved region, designing and testing screening primer probe sequence with high specificity and high sensitivity. The relevant RNA virus information is shown in Table 1.

[0065] Table 1 RNA virus information

[0066]

[0067]

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

[0069] Table 2 Primer and probe sequence information

[0070]

[0071]

[0072] At the same time, the representative viral genome sequence was selected, the sequence fragment was selected according to the viral conservative region designed by the primer and probe, and was constructed into the pRNA-SP6-T7 plasmid vector to obtain the corresponding plasmid. The RNA standard was obtained by using the T7 promoter for RNA transcription.

[0073] The RNA sequence is shown in Table 3.

[0074] Table 3 Synthetic RNA sequence information

[0075]

[0076]

[0077]

[0078]

[0079] Example 2 Multiple qPCR detection method

[0080] The positive RNA standard was selected for primer screening test, and the RNA standard was diluted by 10 times gradient, a total of 7 dilution degrees, diluted to 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 17 different concentrations of standard, and 5 μL of nuclease-free water as negative control template. The qPCR reaction premix was configured as shown in Table 4:

[0081] Table 4 qPCR reaction system

[0082] Component Volume (μl) 2X OneStep RT-PCR Buffer III 10.0 TaKaRa Ex Taq HS (5 U / 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 50X ROX 0.4 RNase-free water 2.8 Standard / sample to be tested 5.0

[0083] ABI7500 PCR instrument, according to Table 5 to set the qPCR reaction program, at 60℃ signal acquisition:

[0084] Table 5 qPCR reaction program

[0085]

[0086]

[0087] Example 3 A multiplex fluorescent 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 6-14, and the standard curves obtained by the primers and probes all meet the acceptable standards (R 2 > 0.99, 90%≤Eff%≤110%, and the recovery rate is between 50%-150%). The results are shown in Tables 6-14.

[0089] Table 6 SFRV standard curve result analysis

[0090]

[0091] UD: undetermined; NA: Not applicable

[0092] Table 7 FHV standard curve result analysis

[0093]

[0094]

[0095] UD: undetermined; NA: Not applicable

[0096] Table 8 BVDV standard curve result analysis

[0097]

[0098]

[0099] UD: undetermined; NA: Not applicable

[0100] Table 9 Analysis of CSFV marker curve results

[0101]

[0102] UD: undetermined; NA: Not applicable

[0103] Table 10 Analysis of JEV marker curve results

[0104]

[0105] UD: undetermined; NA: Not applicable

[0106] Table 11 Analysis of DXV marker curve results

[0107]

[0108]

[0109] UD: undetermined; NA: Not applicable

[0110] Table 12 Analysis of EMCV marker curve results

[0111]

[0112]

[0113] UD: undetermined; NA: Not applicable

[0114] Table 13 Analysis of NωV marker curve results

[0115]

[0116] UD: undetermined; NA: Not applicable

[0117] Table 14 Analysis of SFV marker curve results

[0118]

[0119]

[0120] UD: undetermined; NA: Not applicable

[0121] According to Tables 6-14 and Figure 1 - Figure 18 It is known that the RNA template copy number is selected at 108 ~ 10 2 When the concentration of the virus was within the range, the established fluorescent quantitative PCR detection method had a good amplification curve, and the correlation coefficient R of SFRV was 0.999, and the amplification efficiency was 97.078%; the correlation coefficient R of FHV was 0.999, and the amplification efficiency was 97.078%; the correlation coefficient R of BVDV was 1.000, and the amplification efficiency was 92.881%; the correlation coefficient R of CSFV was 1.000, and the amplification efficiency was 95.910%; the correlation coefficient R of JEV was 0.999, and the amplification efficiency was 92.132%; the correlation coefficient R of DXV was 0.999, and the amplification efficiency was 97.192%; the correlation coefficient R of EMCV was 0.999, and the amplification efficiency was 99.565%; the correlation coefficient R of NωV was 1.000, and the amplification efficiency was 93.150%; the correlation coefficient R of SFV was 1.000, and the amplification efficiency was 92.532%. 2 2 2 2 2 2 2 2 2

[0122] Kit detection limit test of Example 4

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

[0124] Table 15 Analysis of the minimum detection limit results

[0125]

[0126]

[0127] The minimum detection limit results are shown in Table 15. The minimum detection limit standard in this detection method is that all 6 repeats are detected. The minimum detection limit of BVDV is 100 copies / reaction, the minimum detection limit of SFRV, FHV and DXV is 50 copies / reaction, and the minimum detection limit of CSFV, JEV, EMCV, NωV and SFV is 10 copies / reaction.

[0128] ​​​​​​​​​​​Obviously, the above embodiments are merely example for clearly illustrating but not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments need not and can not be enumerated. The obvious changes or variations derived from the above description are still within the protection scope of the present application.

Claims

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

27.

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

3. The test kit of claim 2, wherein: The detection kit comprises positive control, negative control, and fluorescent quantitative PCR reaction reagent.

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

5. The test kit according to claim 3, characterized in that: The positive control comprises a positive RNA standard at a concentration of 2 x 10 8 copies / μL.

6. The test kit of claim 4, wherein: The fluorescent quantitative PCR reaction system in the detection kit is 20 μL, which comprises 15 μL of the fluorescent quantitative PCR reaction premix and 5 μL of a template; the template is RNA of a sample to be detected, a positive control standard and a negative control standard.

7. A multiplex fluorescent quantitative PCR detection method for detecting a plurality of RNA viruses in insect cells for non-diagnostic purposes, characterized in that: The method comprises the following steps: (1) extracting an RNA template of a sample to be detected; (2) establishing a fluorescent quantitative PCR reaction system by using the detection composition according to claim 1; (3) performing fluorescent quantitative PCR detection; (4) analyzing results of the sample to be detected; The plurality of RNA viruses are Sf-rhabdovirus, Flock House virus, bovine viral diarrhea virus, classical swine fever virus, Japanese encephalitis virus, Drosophila X virus, Encephalomyocarditis virus, Nudaurelia capensis ω virus and sandfly fever virus.

8. The method of claim 7, wherein: The fluorescent quantitative PCR reaction system is 20 μL, which comprises 15 μL of the fluorescent quantitative PCR reaction premix and 5 μL of a template; the template is RNA of a sample to be detected, a positive control standard and a negative control standard; the fluorescent quantitative PCR reaction premix comprises 2× One Step RT-PCR Buffer III, Ex Taq HS, RT Enzyme Mix II, ROX Reference Dye internal reference dye, RNase-free water and the detection composition according to claim 1.

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