Multiplex fluorescent quantitative PCR (polymerase chain reaction) detection composition, method and kit for detecting multiple DNA (deoxyribonucleic 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 DNA (deoxyribonucleic acid) viruses in insect cells, and the detection method is non-diagnostic or therapeutic and comprises the following steps: (1) extracting a DNA template of a sample to be detected; (2) establishing a fluorescent quantitative PCR reaction system by adopting the detection composition; (3) carrying out fluorescent quantitative PCR (Polymerase Chain Reaction) detection; and (4) analyzing the result of the sample to be detected. According to the present invention, the primer has strong specificity, the cross reaction does not exist between the primer and the probe, and the probe carries different fluorescence reporter groups so as to meet the simultaneous detection of a variety of DNA viruses in one program. Besides, by repeatedly screening and optimizing a reaction system, including the concentrations of the primers and the probes, the detection method finally needs a very small amount of samples, only 5 [mu] L of nucleic acid samples are needed in each detection, and the nucleic acid samples can still be stably detected.

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 DNA viruses in insect cells. Background Art

[0002] Insect cells are widely used in biomedical fields such as vaccine production, recombinant protein expression, and virus research. For example, Sf9 and Sf21 cells from Spodoptera frugiperda, High Five cells from Trichoplusia ni, and D.mel-2 cells from Drosophila melanogaster. In the production process of biological products, exogenous viruses are considered to be a major safety issue, so it is particularly important to detect the contamination of exogenous viral factors in biological products and even in production cell lines.

[0003] Common DNA viruses that infect insect cells include the Baculoviridae, Vesciviridae, Polydnaviridae, Iridoviridae, Poxviridae, etc. Representative ones include the Autographa californica nuclear polyhedrosis virus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) belonging to the Baculoviridae, the tobacco budworm vesicular virus 3h strain, the black-legged braconid virus, as well as the Aedes aegypti iridovirus, the Chilo suppressalis iridovirus, the mulberry moth poxvirus and the cotton bollworm poxvirus that have been reported to infect insect cells.

[0004] Traditional serological detection methods based on enzyme-linked immunosorbent reaction (ELISA) and PCR detection methods based on nucleic acid analysis have certain limitations. The former requires understanding of the serological characteristics of the virus and specific detection of a certain type of virus or a certain class of virus, while the latter also requires understanding of the viral genome structure, nucleic acid sequence, etc. Most detection methods are cumbersome, time-consuming, and have poor sensitivity and specificity. They can only detect a certain type of virus or a certain class of virus. The detection conditions and procedures of different viruses vary greatly, and cannot meet the needs of simultaneous detection of multiple viruses. Summary of the invention

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

[0006] In order to achieve the above purpose, 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 DNA viruses in insect cells, wherein the detection composition comprises primers and probes for detecting the following DNA viruses:

[0008] Primers and probes for detecting baculovirus: 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 probes for detecting vesicular viruses: 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 probes for detecting polysome DNA viruses: 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] Two sets of primers and probes for detecting iridovirus: 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; and 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;

[0012] Two sets of primers and probes for detecting poxvirus: 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; and 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.

[0013] In a second aspect, the present invention provides a multiplex fluorescent quantitative PCR detection kit for detecting multiple DNA viruses in insect cells, wherein the detection kit comprises the above-mentioned detection composition.

[0014] The probe fluorescent reporter group can be FAM, VIC or Cy5, and the fluorescent quencher group can be BHQ1 or BHQ2.

[0015] In the above technical solution, the detection kit comprises a positive control substance, a negative control substance and a fluorescent quantitative PCR reaction reagent.

[0016] In the above technical solution, the fluorescent quantitative PCR reaction reagent is a fluorescent quantitative PCR reaction premix, including probe method qPCR special reagent Premix Ex Taq, ROX Reference Dye internal reference dye, nuclease-free water, and the above detection composition.

[0017] In the above technical solution, the positive control comprises a positive plasmid standard, and the concentration is 2×10 8 copies / μL.

[0018] 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 DNA of the sample to be tested, the positive control standard and the negative control standard.

[0019] In the above technical solution, the reaction conditions of the reaction system are: pre-denaturation at 95°C for 30s, denaturation at 95°C for 5s, annealing at 60°C for 34s, and 40 cycles.

[0020] In a second aspect, the present invention provides a multiplex fluorescence quantitative PCR detection method for detecting multiple DNA viruses in insect cells, wherein the detection method is not for diagnosis or treatment purposes and comprises the following steps:

[0021] (1) extracting DNA template of the sample to be tested;

[0022] (2) establishing a fluorescent quantitative PCR reaction system using the above detection composition;

[0023] (3) Conducting fluorescence quantitative PCR detection;

[0024] (4) Analysis of test results of samples.

[0025] In the above technical scheme, 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 DNA of the sample to be tested, the positive control standard and the negative control standard; the fluorescent quantitative PCR reaction premix includes the probe method qPCR special reagent Premix Ex Taq, ROX ReferenceDye internal reference dye, nuclease-free water, and the above-mentioned detection composition.

[0026] In the above technical solution, the reaction conditions of the fluorescent quantitative PCR reaction system are: 95°C pre-denaturation for 30s, 95°C denaturation for 5s, 60°C annealing for 34s, and 40 cycles.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention selects conservative sequences of viral genomes when screening and designing primers, which has strong specificity and can detect most subtypes of viruses.

[0029] 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 DNA viruses, and can detect any combination of DNA viruses at the same time.

[0030] 3. By designing multiple pairs of probe primers for the conserved regions of the virus, we finally screened the primer probes with good specificity and high sensitivity, which can accurately quantify 4×10 6 The virus content ranges from copies / μL to 4 copies / μL. The detection sensitivity of all viruses can reach 4 copies / μL, and the minimum sensitivity of some viruses can reach 2 copies / μL.

[0031] 4. The detection kit for detecting multiple DNA viruses in insect cells of the present invention contains positive plasmid standards for detection, and tests the qPCR detection method at the same time. The primers have strong specificity, and there is no cross-reaction between the primer probes. By carrying different fluorescent reporter groups on the probes, it can meet the requirements of detecting multiple DNA viruses simultaneously in one program.

[0032] 5. Through repeated screening and optimization of the reaction system, including the concentrations of primers and probes, the detection method ultimately requires very little sample volume, with a minimum of only 5 μL of nucleic acid sample required for each test and still able to detect stably. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 qPCR amplification curve for BV detection;

[0034] Figure 2 qPCR standard curve for BV detection;

[0035] Figure 3 qPCR amplification curve for HvAV-3h detection;

[0036] Figure 4 qPCR standard curve for HvAV-3h detection;

[0037] Figure 5 qPCR amplification curve for CMBV detection;

[0038] Figure 6 qPCR standard curve for CMBV detection;

[0039] Figure 7 qPCR amplification curve for MIV detection;

[0040] Figure 8qPCR standard curve for MIV detection;

[0041] Fig. 9 qPCR amplification curve for CIV detection;

[0042] Fig.10 qPCR standard curve for CIV detection;

[0043] Fig.11 qPCR amplification curve for AMEV detection;

[0044] Fig.12 qPCR standard curve for AMEV detection;

[0045] Fig.13 qPCR amplification curve for HaEPV detection;

[0046] Fig.14 qPCR standard curve for HaEPV detection. DETAILED DESCRIPTION

[0047] 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 being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0048] 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, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0049] The present invention is now further described in conjunction with specific examples. The following examples are only for explaining the present invention, but do not constitute a limitation of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial channels).

[0050] Experimental materials used in the examples of the present invention:

[0051] Premix and ROX were manufactured by TAKARA, and nuclease-free water was manufactured by Invitrogen;

[0052] DNA extraction kit: purchased from Tiangen, catalog number: DP705;

[0053] The probe qPCR reagent Premix Ex Taq (Probe qPCR) was purchased from TaKaRa (containing ROX Reference Dye internal reference dye), catalog number: RR390A;

[0054] Nuclease-free water was purchased from Invitrogen, catalog number: 10977-015.

[0055] Example 1 Primer probe design and synthesis

[0056] By consulting references and comparing with the NCBI database, the viral genome sequence information was confirmed, representative viral conserved region genome sequences were selected, and primer probes were designed and tested. The relevant insect virus information is shown in Table 1.

[0057] Table 1 DNA virus information

[0058]

[0059] The sequences of primers and probes for fluorescence quantitative PCR detection are shown in Table 2.

[0060] Table 2 Primer probe sequence information

[0061]

[0062] Example 2 Multiplex qPCR Detection Method

[0063] The positive plasmid standard was used to screen the primers and the plasmid standard was diluted 10 times in a gradient, with a total of 7 dilutions, and the dilution was 4×10 6 copies / μL(STD01), 4×10 5 copies / μL(STD02), 4×10 4 copies / μL(STD03), 4×10 3 copies / μL(STD04), 4×10 2 copies / μL(STD05), 4×10 1 There are 7 different concentrations of standard products, 10 copies / μL (STD06) and 4 copies / μL (STD07), and 5 μL of nuclease-free water is taken as a negative control template. The configuration of the qPCR reaction premix is ​​shown in Table 3 below:

[0064] Table 3 qPCR reaction system

[0065] Components Volume (μl) PremixExTaq(ProbeqPCR)(2×Conc.) 10.0 Forward primer (10 μM) 0.4 Reverse primer (10 μM) 0.4 Probe (10 μM) 0.2 50×ROX 0.2 Nuclease-free water 3.8 Standard / test sample 5.0

[0066] Use ABI7500PCR instrument to set up qPCR reaction program according to Table 4 and collect signals at 60°C:

[0067] Table 4 qPCR reaction procedure

[0068]

[0069] Example 3 A multiplex fluorescence quantitative PCR detection kit for detecting multiple DNA viruses in insect cells

[0070] According to the above qPCR detection method, the standard curve results are shown in Tables 5 to 12. The standard curves obtained by the primers and probes all meet the acceptable standards (R 2 >0.99, 90%≤Eff%≤110%, recovery rate between 50%-150%). The results are shown in Tables 5-12.

[0071] Table 5 Analysis of BV curve results

[0072]

[0073]

[0074] UD: undetermined; NA: Not applicable

[0075] Table 6 Analysis of HvAV-3h standard curve results

[0076]

[0077]

[0078] UD: undetermined; NA: Not applicable

[0079] Table 7 Analysis of CMBV curve labeling results

[0080]

[0081] UD: undetermined; NA: Not applicable

[0082] Table 8 MIV curve analysis results

[0083]

[0084]

[0085] UD: undetermined; NA: Not applicable

[0086] Table 9 CIV curve analysis results

[0087]

[0088]

[0089] UD: undetermined; NA: Not applicable

[0090] Table 10AMEV curve analysis results

[0091]

[0092] UD: undetermined; NA: Not applicable

[0093] Table 11 Analysis of HaEPV calibration results

[0094]

[0095] UD: undetermined; NA: Not applicable

[0096] According to Table 5-Table 11 and Figure 1-Figure 14 It can be seen that the template copy number is 2×10 7 When the fluorescence quantitative PCR detection method was established in the range of 20, the BV correlation coefficient R 2 =0.999, the amplification efficiency is 104.084%; the correlation coefficient R 2 =0.999, the amplification efficiency is 101.140%; the correlation coefficient of CMBV is R 2 =0.999, the amplification efficiency is 96.459%; the correlation coefficient of MIV is R 2 =0.999, the amplification efficiency is 100.762%; the correlation coefficient of CIV is R 2 =1.000, the amplification efficiency is 101.132%; the correlation coefficient of AMEV is R 2 =0.999, the amplification efficiency is 99.239%; the correlation coefficient of HaEPV is R 2 =0.998, and the amplification efficiency is 96.756%.

[0097] Example 4 Kit Detection Limit Test

[0098] The detection limit refers to the lowest number of virus copies that can be detected in a sample. Based on the previous primer and probe screening and test results, a detection limit lowering experiment was conducted on the above viruses, testing 2×10 3 copies, 2×102 There were 20 copies, 20 copies and 10 copies / reaction groups, 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 12.

[0099] Table 12 Analysis of minimum detection limit results

[0100]

[0101]

[0102] The results of the minimum detection limit are shown in Table 12. The minimum detection limit standard of this detection method is that all 6 replicates are detected. Among them, the minimum detection limit of HvAV-3h, CMBV, CIV, AMEV, and HaEPV is 20 copies / reaction, and the minimum detection limit of BV and MIV is 10 copies / reaction.

[0103] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A multiplex fluorescence quantitative PCR detection composition for detecting multiple DNA viruses in insect cells, characterized in that: The detection composition includes primers and probes for detecting the following DNA viruses: Primers and probes for detecting baculovirus: 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 vesicular viruses: 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 polysome DNA viruses: 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; Two sets of primers and probes for detecting iridovirus: 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; and 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; Two sets of primers and probes for detecting poxvirus: 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; and 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.

2. A multiplex fluorescence quantitative PCR detection kit for detecting multiple DNA 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 a probe method qPCR special reagent Premix Ex Taq, a 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 plasmid standards, and the concentration is 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 DNA 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: pre-denaturation at 95° C. for 30 s, 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 DNA viruses in insect cells, characterized in that: The detection method is not for diagnosis or treatment purposes and comprises the following steps: (1) extracting DNA 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 the DNA of the sample to be tested, a positive control standard and a negative control standard; the fluorescent quantitative PCR reaction premix includes probe method qPCR special reagent Premix ExTaq, 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: 95° C. pre-denaturation for 30 s, 95° C. denaturation for 5 s, 60° C. annealing for 34 s, and 40 cycles.

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