CN118256658A - Primer and probe combination for detecting various insect viruses, kit and application of kit - Google Patents
Primer and probe combination for detecting various insect viruses, kit and application of kit Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The application provides a primer and probe combination capable of detecting various insect viruses simultaneously, which can effectively detect various insect viruses and variant strains and subtypes thereof, and has strong specificity and high sensitivity. The application also relates to a kit comprising such a combination, and to the use of such a combination and kit for the detection of insect viruses.
Description
Technical Field
The application relates to the fields of biological medicine, fermentation engineering and the like, in particular to the field of detection of insect viruses.
Background
The background description provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present disclosure.
Insect cells are used as main eukaryotic recombinant protein expression systems (insect baculovirus expression systems, baculovirus expression vector system, BVES), are important biological matrixes of protein raw materials for vaccine preparation and target proteins for treatment, and have high application potential. Meanwhile, in the field of gene therapy, along with the wide application of recombinant adeno-associated virus (rAAV), an insect cell virus packaging system using sf9/sf21 also becomes a biological matrix for industrial production which is of great interest. However, the following biosafety problem of insect cells is also an important regulatory project of related biological products and medicines, and detection of exogenous viruses of insects is one of the cores for preventing biosafety events. The exogenous viruses from the insects mainly comprise susceptible viruses of insect cells and arboviruses of high-risk infected people, wherein the susceptible viruses can cause growth retardation or death of the insect cells, and influence the efficiency of related biological fermentation; the latter will deliver the high-risk infectious virus to the user via biological products or drugs produced by the insect cells.
In the existing industry specifications, b.11.2.2 of world health organization technical report series No. 978 (WHO TRS 978) states that for new cell matrices, it is necessary to detect viruses at high risk to humans, such as arboviruses involved in insect cells.
The guidelines of us FDA 2010, "characterization and identification of cell substrates and other biological materials for infectious disease indication virus vaccine production" "(Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications) clearly requires that insect cells require detection of insect viruses of various genera using in vitro methods, as well as PCR methods.
Insect viruses are usually not dead in host insect cells, and some insect cells are inherently infected with low-risk insect viruses, so in vitro methods are generally used for detecting insect viruses. The traditional in vitro method comprises an indication cell method and a transmission electron microscope detection method, wherein the former method needs three indication cell lines, a special cell culture laboratory is matched with the former method, the cell culture time is long, and the latter method needs expensive special equipment, and has high operation requirement and complicated operation process. And in vitro methods can only detect each virus separately, which takes a long time. Thus, there is a need for a solution that is low cost, simple to operate, and capable of detecting multiple insect viruses simultaneously.
Disclosure of Invention
This section presents in simplified form the option of inventive concepts, which will be further apparent from the detailed description below. This section is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
Aiming at the problems in the prior art, the application provides a primer and probe combination for simultaneously detecting a plurality of insect viruses, wherein the primer and probe combination comprises at least any two of the following primer and probe groups:
A first primer and probe set comprising primers shown as SEQ ID NO.1 and SEQ ID NO.2, and a probe shown as SEQ ID NO. 3;
A second primer and probe set comprising primers shown as SEQ ID NO.4 and SEQ ID NO.5, and a probe shown as SEQ ID NO. 6;
A third primer and probe set comprising primers shown as SEQ ID NO.7 and SEQ ID NO.8, and a probe shown as SEQ ID NO. 9;
a fourth primer and probe set comprising primers shown as SEQ ID NO.10 and SEQ ID NO.11, and a probe shown as SEQ ID NO. 12;
a fifth primer and probe set comprising primers shown as SEQ ID NO.13 and SEQ ID NO.14, and a probe shown as SEQ ID NO. 15.
Preferably, the 5 'end of each of SEQ ID No.3, SEQ ID No.6, SEQ ID No.9, SEQ ID No.12, SEQ ID No.15 is modified with a FAM group and the 3' end is modified with an MGB group.
The application also provides a kit for simultaneously detecting a plurality of insect viruses, and the kit comprises a primer and a probe combination according to the principle of the application.
Preferably, the kit comprises a detection well plate provided with a positive reference area, a negative reference area, a sample area and a blank area, wherein three detection wells are arranged for each virus in the positive reference area, the negative reference area, the sample area and the blank area.
Preferably, the detection wells for each virus in the detection well plate contain a first primer and probe set, a second primer and probe set, a third primer and probe set, a fourth primer and probe set, and a fifth primer and probe set, respectively.
Preferably, the positive reference region further comprises a positive control nucleic acid for the target virus in the detection well for each virus.
Preferably, the kit further comprises a PCR reaction liquid component, wherein the PCR reaction liquid component comprises nuclease-free water, quantitative PCR reaction premix, external reference premix and external reference DNA.
Preferably, the external reference premix comprises primers shown as SEQ ID NO.17 and SEQ ID NO.18, and a probe shown as SEQ ID NO.19, and the sequence of the external reference DNA is shown as SEQ ID NO. 16.
Preferably, the 5 'end of SEQ ID NO.19 is modified with HEX groups and the 3' end is modified with MGB groups.
The application also provides a primer and probe combination or application of the kit in detecting insect viruses.
The primer and probe combination, the kit and the application thereof according to the principle of the application can detect various insect viruses and subtypes and variants thereof simultaneously, improve the detection efficiency, and have simple operation and low cost, and solve the problems of single detection type, complex operation and high cost in the prior art.
Drawings
Other or additional features, advantages and details are presented in the following detailed description of the embodiments by way of example only. In the drawings:
FIG. 1 shows the results of performance tests of primer and probe sets against West Nile Virus;
FIG. 2 shows the results of performance testing of primer and probe sets against nodavirus;
FIG. 3 shows the results of performance testing of primer and probe sets against St.Louis encephalitis virus;
FIG. 4 shows the results of performance tests of primer and probe sets against Spodoptera frugiperda rhabdovirus;
FIG. 5 shows the results of performance testing of primer and probe sets for baculoviruses;
FIG. 6 illustrates a detection aperture plate in accordance with the principles of the present application;
FIG. 7 shows the results of detection of West Nile Virus by a kit according to the principles of the present application;
FIG. 8 shows the results of a kit according to the principles of the present application for detecting nodavirus;
FIG. 9 shows the results of detection of St.Louis encephalitis virus by a kit according to the principles of the present application;
FIG. 10 shows the results of a kit according to the principles of the present application for detecting Spodoptera frugiperda rhabdovirus;
FIG. 11 shows the results of a kit according to the principles of the present application for detecting baculovirus; and
FIG. 12 shows a reference DNA amplification curve for a kit according to the principles of the present application.
Detailed Description
The following description is merely exemplary in nature and is not intended to limit the present disclosure, application, or uses. Furthermore, there is no intention to be bound by any expressed or implied theory presented in the preceding technical field, background, and brief summary or the following detailed description. It should be understood that throughout the drawings, corresponding reference numerals indicate like or corresponding parts or features.
The application will now be further elucidated. In the following paragraphs, the different aspects of the application are defined in more detail. Each aspect so defined may be combined with any other aspect(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature(s) indicated as being preferred or advantageous.
In response to the problems of the prior art, the present inventors have summarized common insect viruses for which primer and probe sets for PCR amplification were designed, and information about these common insect viruses is shown in Table 1.
TABLE 1 insect Virus species
| Chinese name | English name | Abbreviations (abbreviations) | Genome type |
| West Nile Virus | West Nile Virus | WNV | ssRNA |
| Nodavirus | Nodamura Virus | NOV | ssRNA |
| St.Louis encephalitis virus | St.Louis encephalitis virus | SLEV | ssRNA |
| Spodoptera frugiperda rhabdovirus | sf-Rhabdoviruses | sfRV | ssRNA |
| Baculovirus | Baculovirus | BacV | dsDNA |
The inventor adopts biological analysis technology to classify and compare all nucleic acid information of insect virus types in Genebank as shown in table 1, extracts a conserved region which can cover the specific insect viruses and variant strains or subtypes thereof to the greatest extent, and uses the conserved region for primer and probe design, thereby designing primer pairs and probe groups for PCR amplification systems for each insect virus, wherein the primers and probes corresponding to each insect virus are shown in table 2:
TABLE 2 primer and Probe set for insect Virus
To verify the detection performance of the above-described primer and probe set for each insect virus, the inventors performed performance tests on the above-described primer and probe set by a PCR amplification procedure in accordance with the requirements of MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments: issuing minimum information for quantitative real-time PCR experiments), wherein the requirements of the performance tests are as follows:
1. The square (R 2) value of the regression coefficient R must be greater than 0.980;
2. the amplification efficiency is in the range of 80% -120% (only for reference);
3. the slope is in the range of-2.8 to-4.0.
If the above requirements are satisfied, it is shown that the primer and probe set have good test performance.
The test experiment method comprises the following steps:
Nucleic acid standards (DNA viruses using artificially synthesized DNA fragments and RNA viruses using in vitro transcribed RNA of artificially synthesized DNA fragments) corresponding to the respective virus detection primers and probe sets were diluted 10-fold in sequence from a concentration of 2X 10 6 copies/mu.l to 6 gradients of 2X 10 6,2×105,2×104,2×103,2×102,2×101, respectively, and then subjected to quantitative PCR reaction at 5. Mu.l to draw a quantitative standard curve. Wherein, the PCR reaction system and the reaction program for DNA viruses and RNA viruses are set as follows:
DNA virus
(1) The quantitative PCR reaction system for each tube of DNA virus is shown in Table 3:
TABLE 3 quantitative PCR reaction System for DNA viruses
| Composition of the components | Volume (mu L) |
| Nuclease-free water | 2.8 |
| Quantitative PCR reaction premix | 10 |
| Upstream primer (10. Mu.M) | 0.5 |
| Downstream primer (10. Mu.M) | 1.5 |
| Probe (5 mu M) | 0.2 |
| Standard substance solution | 5 |
| Total volume of | 20 |
(2) The PCR reaction procedure set up is shown in table 4, with fluorescence channel selection FAM:
TABLE 4 PCR reaction procedure for DNA viruses
RNA Virus
(1) The quantitative PCR reaction system for each tube of RNA virus is shown in Table 5:
TABLE 5 quantitative PCR reaction System for RNA viruses
| Composition of the components | Volume (mu L) |
| Nuclease-free water | 7.8 |
| One-step quantitative PCR reaction premix | 5 |
| Upstream primer (10. Mu.M) | 0.5 |
| Downstream primer (10. Mu.M) | 1.5 |
| Probe (5 mu M) | 0.2 |
| Standard substance solution | 5 |
| Total volume of | 20 |
(2) The PCR reaction procedure set up is shown in table 6, with fluorescence channel selection FAM:
TABLE 6 quantitative PCR reaction System for RNA viruses
3. Amplification curves and standard curves for each insect virus were obtained by the PCR reaction system and reaction procedure described above and are shown in figures 1-5.
4. Lower detection limit
The lower detection limit of each virus is determined by the following method:
(1) Testing 10 copies, 50 copies and 100 copies of nucleic acid standard substances according to each virus detection system, and repeating the test for 3 times for each nucleic acid standard substance;
(2) According to the result of the previous step, selecting the lowest copy number of all 3 repetitions with Ct or Cq value smaller than 38, and testing 24 repetitions; if 23 of the 24 replicates were detectable and CV for Ct or Cq values of all replicates was <20%, then the copy number was confirmed to be the lower limit of detection for the virus detection.
5. By the above experiments, the performance of the probe and primer set for each virus was tested as shown in table 7:
TABLE 7 primer and probe Performance test results for each virus
| Virus abbreviations | Linearity (R 2 value) | Amplification efficiency | Slope of | Detection lower limit (copy number) |
| WNV | 0.998 | 95.2％ | -3.443 | 50 |
| NOV | 0.999 | 87.7％ | -3.656 | 100 |
| SLEV | 0.999 | 90.4％ | -3.575 | 100 |
| sfRV | 0.999 | 84.5％ | -3.760 | 100 |
| BacV | 0.999 | 88.9％ | -3.621 | 50 |
As can be seen from Table 7, the amplification efficiency of the primer and probe set for each insect virus was 80% or more, the linear value (R 2) of the standard curve was greater than 0.980, and the slope of the standard curve was in the range of-2.8 to-4.0, indicating that the primer and probe set according to the principles of the present application were able to effectively detect the corresponding insect virus.
Based on the above experimental results, in one aspect, the present application provides a primer and probe combination capable of simultaneously detecting a plurality of insect viruses, the combination comprising at least two primer and probe sets: (1) A first primer and a probe set for detecting West Nile Virus (WNV), wherein the sequence of the upstream primer is shown as SEQ ID No.1, the sequence of the downstream primer is shown as SEQ ID No.2, and the probe is shown as SEQ ID No. 3; (2) A second primer and a probe set for detecting Nodavirus (NOV), wherein the sequence of the upstream primer is shown as SEQ ID NO.4, the sequence of the downstream primer is shown as SEQ ID NO.5, and the probe is shown as SEQ ID NO. 6; (3) A third primer and a probe set for detecting the St.Louis encephalitis virus (SLEV), wherein the sequence of the upstream primer is shown as SEQ ID NO.7, the sequence of the downstream primer is shown as SEQ ID NO.8, and the probe is shown as SEQ ID NO. 9; (4) A fourth primer and a probe set for detecting spodoptera frugiperda rhabdovirus (sfRV), wherein the sequence of the upstream primer is shown as SEQ ID NO.10, the sequence of the downstream primer is shown as SEQ ID NO.11, and the probe is shown as SEQ ID NO. 12; (5) A fifth primer and a probe set for detecting baculovirus (BacV) are provided, wherein the sequence of the upstream primer is shown as SEQ ID NO.13, the sequence of the downstream primer is shown as SEQ ID NO.14, and the probe is shown as SEQ ID NO. 15. In the above probe sequences SEQ ID No.3, SEQ ID No.6, SEQ ID No.9, SEQ ID No.12 and SEQ ID No.15, the 5 '-end of each sequence was modified with a FAM group and the 3' -end of each sequence was modified with an MGB group.
It will be readily understood by those skilled in the art that in practical applications, the primer and probe set included in the primer and probe combination may be selected according to the number of species of insect virus to be detected, and the corresponding virus is detected by a PCR amplification experiment using the primer and probe set.
In another aspect of the application, there is also provided a kit for simultaneous detection of multiple insect viruses, the kit comprising a primer and probe set for detection of each virus according to the principles of the application. Advantageously, the kit may comprise a detection well plate as shown in fig. 6, which is a 96-well plate and on which a positive reference region, a negative reference region, a sample region and a blank region may be provided to ensure the accuracy of the detection result, each region containing a corresponding primer and probe set in the detection well for each virus. Wherein the sample area is used for detecting whether the insect virus exists in the sample; the positive reference area is used for detecting a positive reference to verify the effectiveness of the experimental system, if the positive reference can be detected, the experimental system is normal, the experiment is effective, otherwise, the experiment is ineffective; the negative reference area is used for detecting a negative reference to verify the specificity of the experiment, if the negative reference is not detected, false positive is not shown, so that the specificity of the experiment is good, otherwise, the specificity is bad; the blank area is used for ensuring that the experimental system is not polluted, and if the blank area does not detect viruses, the experimental system is not polluted, otherwise, the experimental system is polluted. Advantageously, three detection holes are arranged in the positive reference area, the negative reference area, the sample area and the blank area for each virus, so that the situation that a single hole fails to be amplified in a PCR amplification procedure is avoided, the detection efficiency is improved, and the detection error is reduced.
The 96-well plate 100 according to the principles of the present application is set forth in Table 8 below, where 5 viruses are detected using the same pre-filled well plate, and where each of the A-E rows is used to detect one virus. In Table 8, yang Can represents a detection well containing a positive reference, a sample represents a detection well into which the sample is placed, and a negative reference and a blank represent a negative reference well and a blank well, respectively, neither of which contains any control nucleic acid. BacV is DNA virus, but under the one-step reverse transcription quantitative PCR system, bacV detection performance is not affected, and for detection convenience, the same detection system is used for detecting the rest 4 RNA viruses on the same 96-well plate pre-plate.
TABLE 8 detection of functional area distribution of well plates
In each detection well of the detection well plate for each virus, the following substances are preloaded:
(1) All detection wells for each virus contained 5pmol total upstream primer mix, 15pmol total downstream primer mix, and 1pmol total probe mix;
(2) In each detection well for each virus in the positive reference zone, 0.5pg of positive control nucleic acid for the corresponding virus, 500ng of human placental DNA;
(3) The negative reference well, the sample detection well, and the blank detection well do not contain any remaining control nucleic acids.
In alternative embodiments, the assay well plate may be a well plate of other dimensions, such as a 48-well plate or 384-well plate, and may be selected according to the number of virus species to be detected and the specifications of the assay device.
The method for detecting insect viruses using the kit according to the principles of the present application is as follows:
1. sample pretreatment
The collected sample may be pretreated using any viral pretreatment kit. During pretreatment, a negative control substance provided by the detection device is added, namely, human placenta DNA with the final concentration of 100ng/ul is added into a negative control detection hole.
2. Quantitative PCR detection reaction preparation
(1) The reaction solution for one-step reverse transcription quantitative PCR detection was prepared for the detection well plate, and 15. Mu.L of the prepared reaction solution was dispensed into each detection well, and the composition of the reaction solution was as shown in Table 9:
TABLE 9 quantitative PCR detection of reaction solution Components
| Composition of the components | Volume (mu L) |
| Nuclease-free water | 805.6 |
| One-step quantitative PCR reaction premix | 530 |
| External ginseng premix | 212 |
| External reference DNA | 42.4 |
| Total volume of | 1590 |
It will be readily appreciated by those skilled in the art that the total volume of quantitative PCR reaction solution in the assay well plate should be greater than the amount required for use to ensure adequate reaction solution storage during use, and that a portion of the quantitative PCR reaction premix contains the buffers, polymerase and four deoxynucleotides dATP, dGTP, dCTP, dTTP required for the reaction, and that the external reference premix contains the upstream primer, downstream primer and probe for quantitative PCR amplification of the external reference DNA. The kit according to the principle of the present application may include each of the reaction solution components shown in table 9, which are prepared in advance, and the reaction solution components may be directly mixed and prepared for use.
(2) External reference DNA description
The reference DNA (DNA sequences used as external references) shown in table 10 and its corresponding reference premix were used to perform a reference amplification reaction independent of the amplification of the viral sequences of interest. Wherein the external reference premix contains a primer and a probe for specifically detecting a target fragment on the external reference DNA, the probe is marked at the 5 'end by HEX fluorescent groups so as to be different from FAM marking of the virus probe, and the 3' end of the probe is marked by MGB fluorescent groups. Because the fluorescent group amplified by external reference has HEX, the HEX can react with virus amplification in one test well at the same time, and the reaction result can be detected at the same time in different fluorescent channels. The template amount of the reference DNA is fixed so that the detection of the reference fragment in each test well is consistent and can be used to evaluate the homogeneity of the amplification system and whether or not the reaction system or sample has an inhibitor introduced therein, resulting in a false negative result of the detection. The sequence of the reference DNA is shown in Table 10.
TABLE 10 external reference DNA sequences
The sequences of the external reference primers and probes are shown in Table 11:
TABLE 11 sequences of external reference primers and probes
| Name of the name | 5' Modification | Sequence(s) | 3' Modification | Sequence number |
| External reference to the forward direction | GCTATTTCGCGTCTCGTTCTC | 17 | ||
| External parameters reverse direction | GCGTATATGCGCTGCTCTCA | 18 | ||
| External reference probe | HEX | CGCGATTCGTACGCG | MGB | 19 |
3. Loading sample
The loading comprises the following steps:
(1) Adding 5 mu L of matrix solution into each well of a positive reference area of a detection pore plate, wherein the matrix solution comprises eluent, diluent, TE buffer or nuclease-free water after sample pretreatment;
(2) Adding 5 mu L of negative reference substance which is additionally processed in the pretreatment of the sample to each well of the negative reference area of the detection well plate;
(3) Adding 5 mu L of the sample pretreated solution to each well of the sample area of the detection well plate;
(4) Adding 5 mu L of nuclease-free water to each well in the blank area of the detection well plate;
(5) And placing the detection pore plate on a micro-pore plate centrifuge to quickly centrifugally mix the reaction system for 1 minute.
4. Detection reaction
The reaction program shown in Table 12 was set on the quantitative PCR apparatus; the signal acquisition is set in the fourth step, two fluorescent signals are set, one is a detection signal FAM of a virus sequence, and the other is an external reference sequence detection signal HEX.
TABLE 12 quantitative PCR reaction procedure for detection well plate
The assay well plate was placed on a quantitative PCR instrument and the assay was started.
5. Interpretation of results
(1) Experimental effectiveness
The validity of the experiment can be determined according to the following criteria:
a. the standard deviation of Ct or Cq values of all external reference detection on the detection pore plate cannot be larger than 1;
b. the average Ct or Cq value of the positive reference for each virus is less than 25;
c. At least 2 of the 3 duplicate wells for each virus in the negative reference region have a Ct or Cq value greater than 39.9 or N/a;
d. at least 2 of the 3 repeat wells in the blank have a Ct or Cq value greater than 39.9 or N/A.
If the experimental results meet the above 4, the experiment is effective.
(2) Detection result judgment
A. if the average Ct or Cq value for the wells of each virus in the sample area is <35, the corresponding viral nucleic acid is detected as positive;
b. If the average Ct or Cq value of the wells for each virus in the sample area is >35, repeated detection of the virus is required, if the Cq value is still detected
<39.9, The corresponding viral nucleic acid is detected as positive;
c. if the average Ct or Cq value for a well of a virus in the sample region is >39.9 or N/A, then the virus nucleic acid is detected as negative;
6. Detection result
Because of the limitation of virus operation, the inventors replaced viruses with a standard sample for detecting each virus at a lower limit concentration, and tested the performance of the kit by the above method, the amplification curves of the positive reference region, the negative reference region, the sample region and the blank region for each virus were shown in fig. 7 to 11, and the amplification curves of the reference DNA on the assay well plate were shown in fig. 12.
As can be seen from fig. 7-11, within 40 cycles, the positive reference and sample areas exhibited rapidly enhanced fluorescent signals relative to both the negative reference and blank areas, indicating that primer and probe sets and kits according to the principles of the present application were able to effectively detect the corresponding insect viruses. Wherein, the rapid enhanced fluorescence signal in the positive reference area indicates that the detection experiment is effective, and the reagent, the instrument and the like all run normally; the negative reference area has no rapid enhanced fluorescence signal, which indicates that false positive does not occur in the detection process, and further indicates that the primer and the probe set kit according to the principle of the application have good specificity; the blank areas have no fluorescence signal enhancement, which indicates that the experimental environment is uncontaminated and the experimental result is effective. As can be seen from the amplification curves of FIG. 12, the reference DNA amplification curves of the different wells on each assay well plate have substantially the same shape within 40 cycles, indicating good uniformity of the amplification system and absence of inhibitors in the reaction system. The detection result shows that the primer and probe combination and the kit have good detection effect.
While at least one exemplary embodiment has been presented in the foregoing detailed description, it should be appreciated that a vast number of variations exist. It should also be appreciated that the exemplary embodiment or exemplary embodiments described herein are only examples, and are not intended to limit the scope, applicability, or configuration of the application in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing the exemplary embodiment or exemplary embodiments. It should be understood that various changes, modifications, or alterations can be made in the function and arrangement of elements without departing from the scope of the application as set forth in the appended claims and the equivalents thereof.
Claims (10)
1. A primer and probe combination for simultaneously detecting a plurality of insect viruses, wherein the primer and probe combination comprises at least any two of the following primer and probe sets:
A first primer and probe set comprising primers shown as SEQ ID NO.1 and SEQ ID NO.2, and a probe shown as SEQ ID NO. 3;
A second primer and probe set comprising primers shown as SEQ ID NO.4 and SEQ ID NO.5, and a probe shown as SEQ ID NO. 6;
A third primer and probe set comprising primers shown as SEQ ID NO.7 and SEQ ID NO.8, and a probe shown as SEQ ID NO. 9;
a fourth primer and probe set comprising primers shown as SEQ ID NO.10 and SEQ ID NO.11, and a probe shown as SEQ ID NO. 12;
And a fifth primer and probe set comprising primers shown as SEQ ID NO.13 and SEQ ID NO.14, and a probe shown as SEQ ID NO. 15.
2. The primer and probe combination of claim 1, wherein each of the 5 'ends of SEQ ID No.3, SEQ ID No.6, SEQ ID No.9, SEQ ID No.12, SEQ ID No.15 is modified with a FAM group and each of the 3' ends is modified with an MGB group.
3. A kit for simultaneous detection of a plurality of insect viruses, comprising a primer and probe combination according to claim 1 or 2.
4. A kit according to claim 3, comprising a detection well plate provided with a positive reference area, a negative reference area, a sample area and a blank area, wherein three detection wells are provided for each virus in the positive reference area, the negative reference area, the sample area and the blank area.
5. The kit according to claim 4, wherein the detection wells for each virus in the detection well plate comprise a first primer and probe set, a second primer and probe set, a third primer and probe set, a fourth primer and probe set, and a fifth primer and probe set, respectively.
6. The kit of claim 5, wherein the positive reference zone further comprises a positive control nucleic acid for the target virus in a detection well for each virus.
7. The kit of claim 6, further comprising PCR reaction solution components comprising nuclease-free water, quantitative PCR reaction premix, external reference premix, and external reference DNA.
8. The kit of claim 7, wherein the external reference premix comprises primers shown as SEQ ID NO.17 and SEQ ID NO.18, and a probe shown as SEQ ID NO.19, and the external reference DNA has a sequence shown as SEQ ID NO. 16.
9. The kit of claim 8, wherein the 5 'end of SEQ ID No.19 is modified with a HEX group and the 3' end is modified with an MGB group.
10. Use of a primer and probe combination as claimed in claim 1 or 2, or a kit as claimed in any one of claims 3 to 9, for the detection of insect viruses.
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