CN112266978A - Primer-probe combination, detection kit and application thereof - Google Patents
Primer-probe combination, detection kit and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a primer probe combination, a detection kit and application thereof. According to the invention, the first Y primer and the second Y primer are designed, the first Y primer can be partially complementary with the specific primer of the dengue virus, the second Y primer can be partially complementary with the specific primer of the Zika virus to form a Y-shaped structure, so that the initiation of a non-specific extension reaction is prevented, the problem of insufficient detection specificity caused by higher similarity of genome sequences of the dengue virus and the Zika virus is avoided, the specificity and the accuracy in the detection process of the dengue virus and/or the Zika virus are improved, and the application prospect and the market value are good.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a primer probe combination, a detection kit and application thereof, and a detection method of dengue virus and/or Zika virus.
Background
Dengue virus (Dengue virus) belongs to the flavivirus family of Flaviviridae, is a single positive strand RNA virus, Zika virus (Zika virus) belongs to the flavivirus family of Flaviviridae, has an envelope, and has a single positive strand RNA genome.
At present, the methods for diagnosing dengue virus and Zika virus at home and abroad mainly comprise serological tests, virus culture and PCR. Wherein, serological test and virus culture have the defects of low sensitivity, immunological cross reaction, long period and the like; most of the existing in-vitro diagnosis kits for dengue virus and Zika virus adopt a single RT-PCR reaction mode, and the problems of low detection flux and long time consumption exist in the process of large-scale sample detection in the epidemic season of the entomovirus. In addition, the conventional multiplex real-time fluorescence PCR technology is a technology combining the PCR technology and a multicolor fluorescence labeling probe, can simultaneously and rapidly amplify a plurality of viruses in the same PCR reaction tube, and greatly improves the detection efficiency. Although the method has the characteristics of high speed, sensitivity, high automation degree and the like, pathogens with high intergeneric similarity, such as dengue viruses and Zika viruses with high genome sequence similarity, have the problems of insufficient specificity and easy occurrence of cross reaction, and further influence the specificity and accuracy of the detection of the kit. Therefore, it is necessary to establish a more accurate and efficient dual detection method capable of simultaneously detecting dengue virus and Zika virus.
Disclosure of Invention
The invention aims to provide a primer probe combination, a detection kit and application thereof, and a detection method of dengue viruses and/or Zika viruses, and aims to solve the technical problems of low specificity and accuracy in the existing detection process of the dengue viruses and/or the Zika viruses.
In order to achieve the above objects, in one aspect, the present invention provides a primer probe combination, which includes a first primer probe set and a second primer probe set, wherein the first primer probe set includes a dengue virus specific primer, a first Y primer and a first fluorescent probe, and the first Y primer includes at least one of a first Y primer and a first second Y primer; the second type of primer probe group comprises Zika virus specific primers, a second type of Y primers and a second type of fluorescent probes, and the second type of Y primers comprise at least one of a second type of first Y primers and a second type of second Y primers;
wherein, the nucleotide sequence of the dengue virus specific primer is the nucleotide sequence shown in SEQ ID NO. 1-2, or the nucleotide sequence with the same function obtained by deletion, insertion or replacement of the nucleotide sequence shown in SEQ ID NO. 1-2;
the nucleotide sequence of the first Y primer is the nucleotide sequence shown in SEQ ID NO. 3, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 3;
the nucleotide sequence of the first type second Y primer is the nucleotide sequence shown in SEQ ID NO. 4, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 4;
the nucleotide sequence of the first type of fluorescent probe is the nucleotide sequence shown in SEQ ID NO. 5, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 5;
the nucleotide sequence of the Zika virus specific primer is the nucleotide sequence shown in SEQ ID NO. 6-7, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 6-7;
the nucleotide sequence of the second type first Y primer is the nucleotide sequence shown in SEQ ID NO. 8, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 8;
the nucleotide sequence of the second type second Y primer is the nucleotide sequence shown in SEQ ID NO. 9, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 9;
the nucleotide sequence of the second fluorescent probe is a nucleotide sequence shown in SEQ ID NO. 10, or a nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 10;
the fluorophore on the first type of fluorescent probe is different from the fluorophore on the second type of fluorescent probe.
In another aspect of the present invention, a detection kit is provided, which includes the primer probe combination provided by the present invention.
In another aspect of the invention, the application of the detection kit in the detection of dengue virus and/or Zika virus is provided.
The primer probe combination provided by the invention designs a first Y primer and a second Y primer, wherein the first Y primer can be partially complementary with a specific primer of dengue virus, and the second Y primer can be partially complementary with a specific primer of Zika virus to form a Y-shaped structure. When the 3 'end of the specific primer of the dengue virus and/or Zika virus is identified and hybridized with the target sequence, the 5' end is separated from the first type Y primer and/or the second type Y primer, and an extension reaction is started; when no target sequence exists or the sequence of the sample to be detected is not completely matched with the 5' end sequence, the first Y primer and the specific primer of the dengue virus, and the second Y primer and the specific primer of the Zika virus continuously keep a Y-shaped structure, so that the extension reaction can not be started. The primer probe combination provided by the invention can avoid the problem of insufficient detection specificity caused by higher similarity of genome sequences of the dengue virus and the Zika virus, and avoid non-specific amplification, thereby improving the specificity and accuracy in the detection process of the dengue virus and/or the Zika virus.
The detection kit provided by the invention comprises the primer probe combination provided by the invention, so that the specificity, the accuracy and the sensitivity to the dengue virus and/or Zika virus are higher in detection, and the detection result can be used for research in the fields of conventional monitoring of the dengue virus and/or Zika virus and the like. Meanwhile, the detection kit provided by the invention can identify the infection conditions of the sample to be detected on the dengue virus and the Zika virus only by detecting one sample to be detected once, the detection process only needs 1.5 hours, the detection time can be saved, the quantity of materials to be obtained can be reduced, and the detection efficiency can be obviously improved.
The detection kit provided by the invention comprises the primer probe combination provided by the invention, so that the detection kit can be used for independently and rapidly detecting the dengue virus or Zika virus during detection, can avoid the problem of insufficient detection specificity caused by higher similarity of genome sequences of the dengue virus and the Zika virus, realizes double rapid detection of the dengue virus and the Zika virus, and has the advantages of strong specificity, good accuracy and high sensitivity. The detection process of the detection kit provided by the invention only needs 1.5 hours, the detection time can be obviously shortened, the detection efficiency is improved, and the detection kit has good application prospect and market value.
Drawings
FIG. 1 is a schematic diagram of the process of performing match amplification of a first type of first Y primer and a dengue virus specific primer provided by the present invention with a sequence of a target sequence;
FIG. 2 is a schematic diagram of the process that the first type of first Y primer and the dengue virus specific primer provided by the present invention cannot be matched and amplified with the sample to be detected (non-target sequence);
FIG. 3 is an amplification curve obtained by detecting a positive quality control substance in example 2 of the present invention;
FIG. 4 is an amplification curve obtained by detecting a negative quality control substance in example 3 of the present invention.
FIG. 5 is an amplification curve obtained by detecting dengue virus in example 4 of the present invention;
FIG. 6 is an amplification curve obtained by assaying Zika virus samples according to example 5 of the present invention;
FIG. 7 is an amplification curve obtained by detecting a mixed sample of dengue virus and Zika virus in example 6 of the present invention.
Detailed Description
In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and the embodiments described below are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention. Those whose specific conditions are not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the description of the present invention, the term "and/or" describing an association relationship of associated objects means that there may be three relationships, for example, a and/or B, may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
It should be understood that the weight of the related components mentioned in the embodiments of the present invention may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, it is within the scope of the disclosure that the content of the related components is scaled up or down according to the embodiments of the present invention. Specifically, the weight described in the embodiments of the present invention may be a unit of mass known in the chemical field such as μ g, mg, g, kg, etc.
In addition, unless the context clearly uses otherwise, an expression of a word in the singular is to be understood as including the plural of the word. The terms "comprises" or "comprising" are intended to specify the presence of stated features, quantities, steps, operations, elements, portions, or combinations thereof, but are not intended to preclude the presence or addition of one or more other features, quantities, steps, operations, elements, portions, or combinations thereof.
It should be noted that the molecular biology experimental methods not specifically described in the examples of the present invention are performed by referring to the specific methods listed in the molecular cloning experimental manual (third edition) j. sambrook, or according to the kit and the product specification; related reagents and biomaterials, if not specifically stated, are commercially available.
The embodiment of the invention provides a primer probe combination, which comprises a first primer probe group and a second primer probe group, wherein the first primer probe group comprises a dengue virus specific primer, a first Y primer and a first fluorescent probe, and the first Y primer comprises at least one of a first Y primer and a first second Y primer; the second type of primer probe group comprises a Zika virus specific primer, a second type of Y primer and a second type of fluorescent probe, and the second type of Y primer comprises at least one of a second type of first Y primer and a second type of second Y primer;
wherein, the nucleotide sequence of the dengue virus specific primer is the nucleotide sequence shown in SEQ ID NO. 1-2, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 1-2;
the nucleotide sequence of the first Y primer is the nucleotide sequence shown in SEQ ID NO. 3, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 3;
the nucleotide sequence of the first type second Y primer is the nucleotide sequence shown in SEQ ID NO. 4, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 4;
the nucleotide sequence of the first type of fluorescent probe is the nucleotide sequence shown in SEQ ID NO. 5, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 5;
the nucleotide sequence of the Zika virus specific primer is the nucleotide sequence shown in SEQ ID NO. 6-7, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 6-7;
the nucleotide sequence of the second type first Y primer is the nucleotide sequence shown in SEQ ID NO. 8, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 8;
the nucleotide sequence of the second type of second Y primer is the nucleotide sequence shown in SEQ ID NO. 9, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 9;
the nucleotide sequence of the second fluorescent probe is the nucleotide sequence shown in SEQ ID NO. 10, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 10;
the fluorophore on the first type of fluorescent probe is different from the fluorophore on the second type of fluorescent probe.
Nucleotide sequences of dengue virus-specific primers (DEN-FP and DEN-RP) (SEQ ID NOS: 1-2):
DEN-FP：GACTAGAGGTTAGAGGAGAC
DEN-RP：AGACAGCAGGATCTCTGGT
nucleotide sequence of first type first Y primer DEN-FP2 (SEQ ID NO: 3):
DEN-FP 2: ACTGTTCCGAACCTCTAGTC-P ("P" means the 3' end is blocked with phosphate)
Nucleotide sequence of first type second Y primer DEN-RP2 (SEQ ID NO: 4):
DEN-RP 2: GTTGACTGTACTGCTGTCT-P ("P" means the 3' end is blocked with phosphate)
Nucleotide sequence of the first type of fluorescent probe (SEQ ID NO: 5):
CTYTCCCAGCGTCAATATGCTGTTTGT
nucleotide sequences of Zika virus-specific primers (ZIKA-FP and ZIKA-RP) (SEQ ID NOS: 6-7):
ZIKA-FP：TGGAGATGAYTGCGTTGTGAAR
ZIKA-RP：TTCCTAACTTTTCCCATRTCATTCAA
the nucleotide sequence of the second type of first Y primer ZIKA-FP2 (SEQ ID NO: 8):
ZIKA-FP 2: GCCACACCCGCARTCATCTCCA-P ("P" means the 3' end is blocked with phosphate)
The nucleotide sequence of the second type of second Y primer ZIKA-RP2 (SEQ ID NO: 9):
ZIKA-RP 2: CCACCCACYATGGGAAAAGTTAGGAA-P ("P" means the 3' end is blocked with phosphate)
Nucleotide sequence of the second type of fluorescent probe (SEQ ID NO: 10):
CAATYGATGATAGGTTTGCACATGCCCTC
in the primer probe combination provided by the embodiment of the invention, a first Y primer and a second Y primer are designed, wherein the first Y primer can be partially complementary with a specific primer of dengue virus, and the second Y primer can be partially complementary with a specific primer of Zika virus to form a Y-shaped structure. When the 3 'end of the specific primer of the dengue virus and/or Zika virus is identified and hybridized with the target sequence, the 5' end is separated from the first type Y primer and/or the second type Y primer, and an extension reaction is started; when no target sequence exists or the sequence of the sample to be detected is not completely matched with the 5' end sequence, the first Y primer and the specific primer of the dengue virus, and the second Y primer and the specific primer of the Zika virus continuously keep a Y-shaped structure, so that the extension reaction can not be started. The primer probe combination provided by the embodiment of the invention can avoid the problem of insufficient detection specificity caused by higher similarity of genome sequences of the dengue virus and the Zika virus, and avoid non-specific amplification, thereby improving the specificity and accuracy in the detection process of the dengue virus and/or the Zika virus.
Specifically, in the first primer probe group, the first Y primer of the first type can be complementary with the 5' end part of the dengue virus specific primer DEN-FP to form a Y-shaped structure; the first second Y primer can be complementary to the 5' end of the dengue virus specific primer DEN-RP to form a "Y" type structure. Wherein the first fluorescent probe can be combined with one of the sense strand and the antisense strand of the dengue virus target sequence. Taking the first type of first Y primer as an example, the matching process between the Y primer and the specific primer is specifically described below with reference to FIGS. 1 and 2: in FIG. 1, a first Y primer of a first type is complementary to a 5 ' end portion of a dengue virus specific primer DEN-FP to form a "Y" type structure, and in a detection process, when a 3 ' end of the DEN-FP is hybridized with a target sequence in an identification manner, the 5 ' end of the DEN-FP is separated from the first Y primer, and at this time, an extension reaction is initiated to separate a fluorescent group and a quenching group on a first fluorescent probe, so that a fluorescent detection system can receive a fluorescent signal emitted by the fluorescent group to detect the target sequence, wherein the target sequence is a dengue virus positive sequence; in fig. 2, when no target sequence exists or the sequence of the sample to be detected is not completely matched with the DEN-FP, the 5' end of the DEN-FP and the first type of first Y primer continue to maintain a "Y" type structure, at this time, an extension reaction cannot be started, a fluorescent signal emitted by a fluorescent group on the first type of fluorescent probe is continuously absorbed by a quencher group, a fluorescent detection system cannot receive the fluorescent signal emitted by the fluorescent group, and the sample to be detected is dengue virus negative. Similarly, the first second Y primer is complementary with the 5 ' end part of the dengue virus specific primer DEN-RP to form a Y-shaped structure, when the 3 ' end of the DEN-RP is identified and hybridized with a target sequence in the detection process, the 5 ' end of the DEN-RP is separated from the first second Y primer, and at the moment, an extension reaction is started to separate a fluorescent group and a quenching group on the first fluorescent probe, so that a fluorescent detection system can receive a fluorescent signal emitted by the fluorescent group to realize the detection of the target sequence, and the target sequence is a dengue virus positive sequence; when no target sequence exists or the sequence of the sample to be detected is not completely matched with the DEN-RP, the 5' end of the DEN-RP and the first-class second Y primer continue to keep a Y-shaped structure, at the moment, the extension reaction cannot be started, the fluorescent signal emitted by the fluorescent group on the first-class fluorescent probe is continuously absorbed by the quenching group, the fluorescent detection system cannot receive the fluorescent signal emitted by the fluorescent group, and the sample to be detected is dengue virus negative. In some embodiments, the first type first Y primer and the first type second Y primer are preferably combined with the dengue virus specific primer to form a "Y" structure, and when the first type primer probe set is used for detecting dengue virus, the detection specificity can be further improved.
Correspondingly, in the second type of primer probe set, the second type of first Y primer can be complementary with the 5' end part of the Zika virus specific primer ZIKA-FP to form a Y-shaped structure; the second type of second Y primer can be complementary to the 5' end portion of the Zika-RP, which is a Zika virus-specific primer, to form a "Y" type structure. Wherein the second type of fluorescent probe can bind to one of the sense strand and the antisense strand of the Zika virus target sequence. The identification process of the second type of first Y primer and the Zika virus specific primer ZIKA-FP, the identification process of the second type of second Y primer and the Zika virus specific primer ZIKA-RP in the detection process and the fluorescence response process of the second type of fluorescent probe are similar to the process of the first type of primer probe group, and the description is omitted here. In some embodiments, the second type of first Y primer and the second type of second Y primer are preferably combined with the zika virus-specific primers to form a "Y" structure, and when the second type of primer probe set is used for detecting zika virus, the detection specificity can be further improved.
It is understood that, in order to simultaneously detect dengue virus and Zika virus, the fluorescence forms of the first type of fluorescent probe and the second type of fluorescent probe need to be distinguished, and thus the fluorophore on the first type of fluorescent probe is different from the fluorophore on the second type of fluorescent probe. In some embodiments, the first type of fluorescent probe has a fluorophore FAM at the 5 'end, a quencher BHQ1 at the 3' end, a fluorophore HEX at the 5 'end, and a quencher BHQ2 at the 3' end. At this time, the first type of fluorescent probe is named as DENV-FAM, the second type of fluorescent probe is named as ZIKA-HEX, and the sequences are respectively as follows:
FAM-CTYTCCCAGCGTCAATATGCTGTTTGT-BHQ1
HEX-CAATYGATGATAGGTTTGCACATGCCCTC-BHQ2
the embodiment of the invention also provides a detection kit, which comprises the primer probe combination provided by the embodiment of the invention.
The detection kit provided by the embodiment of the invention comprises the primer probe combination provided by the embodiment of the invention, so that the specificity, the accuracy and the sensitivity to the dengue virus and/or Zika virus are higher in detection, and the detection result can be used for research in the fields of conventional monitoring of the dengue virus and/or Zika virus and the like. Meanwhile, the detection kit provided by the embodiment of the invention can identify the infection conditions of the sample to be detected on the dengue virus and the Zika virus only by detecting one sample to be detected once, the detection process only needs 1.5 hours, the detection time can be saved, the material drawing quantity can be reduced, and the detection efficiency can be obviously improved.
In some embodiments, the final concentration of dengue virus-specific primers is 0.50 μ M to 1.0 μ M (0.25 μ M to 0.50 μ M for DEN-FP and DEN-RP, respectively), the final concentration of the first type of Y primers is 0.50 μ M to 1.0 μ M (0.25 μ M to 0.50 μ M for DEN-FP2 and DEN-RP2, respectively), and the final concentration of the first type of probes is 0.10 μ M to 0.30 μ M; the final concentration of the Zika virus specific primers was 0.50. mu.M-1.0. mu.M (0.25. mu.M-0.50. mu.M for ZIKA-FP and ZIKA-RP, respectively), the final concentration of the second type of Y primers was 0.50. mu.M-1.0. mu.M (0.25. mu.M-0.50. mu.M for ZIKA-FP2 and ZIKA-RP2, respectively), and the final concentration of the second type of probe was 0.10. mu.M-0.30. mu.M.
In some embodiments, the test kit further comprises at least one of a reaction buffer, an enzyme mixture, and a quality control. In some embodiments, the reaction system of the assay kit is a 25 μ L reaction system, the components of which are shown in Table 1. It is understood that the total number of reaction systems of the detection kit can also be adjusted according to actual conditions, and the concentrations of the components are adjusted according to proportions.
TABLE 125 μ L reaction composition of the reaction System
The embodiment of the invention also provides application of the detection kit in detecting dengue viruses and/or Zika viruses.
The detection kit provided by the embodiment of the invention comprises the primer probe combination provided by the embodiment of the invention, so that the detection kit can be used for independently and rapidly detecting the dengue virus or the Zika virus during detection, can avoid the problem of insufficient detection specificity caused by higher similarity of genome sequences of the dengue virus and the Zika virus, realizes dual rapid detection of the dengue virus and the Zika virus, and has the advantages of strong specificity, good accuracy and high sensitivity. The detection process of the detection kit provided by the embodiment of the invention only needs 1.5 hours, can obviously shorten the detection time and improve the detection efficiency, and has good application prospect and market value.
Specifically, when the detection kit provided by the embodiment of the invention is used for detecting dengue virus and/or Zika virus, the method comprises the following steps:
s1, providing total RNA of the sample to be detected;
s2, carrying out RT-qPCR amplification on the total RNA by using a detection kit to obtain a fluorescence curve;
s3, when the fluorescence curve is S-shaped and the Ct value is less than or equal to 38, the result is positive; when the fluorescence curve is S-shaped and the Ct value is more than 38 and less than 40, the result is suspicious, and the RT-qPCR amplification is carried out on the total RNA again; negative results were obtained when the fluorescence curve was non-sigmoidal and the Ct value was 40 or no Ct value.
Specifically, in S1, the total RNA extraction method for the test sample may be a method that is conventional in the art.
In S2, the method for performing RT-qPCR amplification on total RNA in the sample to be tested may be a method conventional in the art. In some embodiments, the reaction procedure of RT-qPCR amplification in a 25 μ L reaction system is optimized to improve amplification efficiency and detection efficiency, and specifically includes three steps of reverse transcription, denaturation and amplification, and the reaction conditions of each step are shown in table 2.
TABLE 2 reaction procedure for RT-qPCR
In S3, the detection result of the sample to be detected can be obtained by combining the Ct value range according to the difference of the obtained fluorescence curves. In Ct values, C represents Cycle and t represents threshold, which means: the number of cycles that the fluorescence signal in each reaction tube undergoes when it reaches a set threshold. When the fluorescence curve is S-shaped and the Ct value is less than or equal to 38, the sample to be detected is positive, and the dengue virus/Zika virus of the sample to be detected can be determined to be positive according to the fluorescence source emitted by the sample to be detected. For example, when the fluorophore on the first type of fluorescent probe is FAM and the fluorophore on the second type of fluorescent probe is HEX, the dengue virus is positive when the fluorescence signal emitted by the detection result is FAM; when the fluorescence signal emitted by the detection result is HEX, the Zika virus is positive; when the fluorescence signal emitted by the detection result is the mixture of FAM and HEX, the dengue virus and the Zika virus are both positive.
When the fluorescence curve is S-shaped and the Ct value is more than 38 and less than 40, the fluorescence curve is a suspicious result, the RT-qPCR amplification is carried out on the total RNA again, if the double-check result shows that the Ct value is less than 40 and the fluorescence curve is S-shaped, the sample to be detected is positive, and then the sample to be detected is confirmed to be positive specifically dengue virus and/or Zika virus according to the fluorescence signal emitted by the sample to be detected; if the rechecking result shows that the Ct value is more than or equal to 40 or the fluorescence curve is not S-shaped, the sample to be detected is negative.
When the fluorescence curve is non-S type and the Ct value is 40 or no Ct value, the sample to be tested is negative.
Through the detection steps, the method can be used for detecting the dengue virus and the Zika virus independently, can realize double detection of the dengue virus and the Zika virus, and can avoid the problem of insufficient specificity caused by high similarity of genome sequences of the dengue virus and the Zika virus in the presence of the first primer probe group and the second primer probe group.
In order to make the above implementation details and operations of the present invention clearly understood by those skilled in the art and to make the progress of the primer probe combination, the detection kit and the application thereof obviously apparent, the above technical solutions are illustrated by the following embodiments.
Example 1
The embodiment provides a detection kit, which comprises an RT-PCR reaction solution, an RT-PCR enzyme mixed solution, a positive quality control product and a negative quality control product.
The RT-PCR reaction solution comprises 2 xOne-Step RT-qPCR Buffer, dengue virus specific primers (DENV-FP and DENV-RP), first Y primers (DENV-FP2 and DENV-RP2), first fluorescent probes (DENV-FAM), Zika virus specific primers (ZIKA-FP and ZIKA-RP), second Y primers (ZIKA-FP2 and ZIKA-RP2) and second fluorescent probes (ZIKA-HEX), wherein the primers and the fluorescent probes are obtained by the following steps: all known dengue and Zika virus whole genome sequences were downloaded from NCBI, sequence alignment was performed using Clone Manager software to find specific conserved regions for each type of sequence, and then Primer Express 3.0 software was used to design dengue virus specific primers (DENV-FP, DENV-RP), first type Y Primer Y primers (DENV-FP2, DENV-RP2) and first type fluorescent probes (DENV-FAM), Zika virus specific primers (ZIKA-FP, ZIKA-RP), second type Y primers (ZIKA-FP2, ZIKA-RP2) and second type fluorescent probes (ZIKA-HEX). All primers and probes were synthesized by Bionics (Shanghai) GmbH; the 2 XOne-Step RT-qPCR Buffer and the enzyme mixed liquor are both from Quanti Probe One-Step RT-qPCR Kit (Shenzhen Union medicine science and technology Co., Ltd.).
The positive quality control product is a synthetic plasmid mixture containing target gene segments of the dengue virus and the Zika virus, and the negative quality control product is DEPC water.
The reaction system of the detection kit provided in this example is 25. mu.L, and the composition thereof is shown in Table 1.
TABLE 125 μ L reaction composition of the reaction System
Example 2
In this embodiment, the detection kit obtained in example 1 is used to detect a positive quality control substance, and includes the following steps:
(11) and (3) positive quality control product verification: taking a positive quality control product in the reagent kit for verification; program setting is carried out by using an ABI7500 type full-automatic fluorescent quantitative PCR detector, and fluorescent reporter groups are as follows: FAM, VIC channel, Passive Reference: NONE, reaction procedure as shown in table 2;
(12) after the amplification was completed, the fluorescence curve obtained is shown in FIG. 3.
As can be seen from FIG. 3, both the two fluorescence curves are S-shaped, Ct values are both less than or equal to 38, and the first type of fluorescent probe and the second type of fluorescent probe both emit light, which indicates that the detection kit obtained in example 1 can simultaneously detect dengue virus (DEN) and Zika virus (ZIKA).
TABLE 2 reaction procedure for RT-qPCR
Example 3
In this embodiment, the detection kit obtained in example 1 is used to detect a negative quality control material, and includes the following steps:
(21) and (3) verifying negative quality control products: taking DEPC water for negative quality control product verification; program setting is carried out by using an ABI7500 type full-automatic fluorescent quantitative PCR detector, and fluorescent reporter groups are as follows: FAM, VIC channel, Passive Reference: NONE, reaction procedure as shown in table 2;
(22) after the amplification was completed, the fluorescence curve obtained is shown in FIG. 4.
As can be seen from FIG. 4, when the detection kit obtained in example 1 is used for detecting a negative quality control product, the obtained fluorescence curve is non-S-shaped and has no Ct value, which indicates that dengue virus and Zika virus do not exist, the detection result is accurate, and false positive does not occur.
Example 4
(31) Sample preparation: collecting dengue virus 1 strains;
(32) extracting nucleic acid of a sample: nucleic acid extraction was performed on the samples according to the QIAamp Viral RNA Kit method. The positive quality control substance and the negative quality control substance can be directly used without extraction;
(33) program setting is carried out by using an ABI7500 type full-automatic fluorescent quantitative PCR detector, and fluorescent reporter groups are as follows: FAM, VIC channel, Passive Reference: NONE, reaction procedure as shown in table 2;
(34) after the amplification was completed, the fluorescence curve obtained is shown in FIG. 5.
As can be seen from FIG. 5, when the detection kit obtained in example 1 detects a dengue virus sample, the obtained fluorescence curve is S-shaped, the Ct value is less than or equal to 38, and only the fluorescence signal of the first type of fluorescent probe DENV-FAM is collected, which indicates dengue virus positive and Zika virus negative.
Example 5
(41) Sample preparation: collecting Zika virus 1 strain;
(42) extracting nucleic acid of a sample: nucleic acid extraction was performed on the samples according to the QIAamp Viral RNA Kit method. The positive quality control substance and the negative quality control substance can be directly used without extraction;
(43) program setting is carried out by using an ABI7500 type full-automatic fluorescent quantitative PCR detector, and fluorescent reporter groups are as follows: FAM, VIC channel, Passive Reference: NONE, reaction procedure as shown in table 2;
(44) after the amplification was completed, the fluorescence curve obtained is shown in FIG. 6.
As can be seen from FIG. 6, when the detection kit obtained in example 1 was used to detect Zika virus samples, the fluorescence curve obtained was S-shaped, Ct value was not more than 38, and only the fluorescence signal of the second type of fluorescent probe ZIKA-HEX was collected, indicating dengue virus negative and Zika virus positive.
Example 6
(51) Sample preparation: 1 strain of each of dengue virus and Zika virus is collected;
(52) extracting nucleic acid of a sample: nucleic acid extraction was performed on the samples according to the QIAamp Viral RNA Kit method. The positive quality control substance and the negative quality control substance can be directly used without extraction;
(53) program setting is carried out by using an ABI7500 type full-automatic fluorescent quantitative PCR detector, and fluorescent reporter groups are as follows: FAM, VIC channel, Passive Reference: NONE, reaction procedure as shown in table 2;
(54) after the amplification was completed, the fluorescence curve obtained is shown in FIG. 7.
As can be seen from FIG. 7, when the detection kit obtained in example 1 detects a dengue virus and Zika virus mixed sample, two fluorescence curves are obtained, both of which are S-shaped, Ct values are not more than 38, and fluorescence signals of a first type of fluorescent probe DENV-FAM and a second type of fluorescent probe ZIKA-HEX can be simultaneously collected, which indicates that the dengue virus and the Zika virus are both positive.
Example 7
Dengue virus concentration sample (1X 10) using the detection kit obtained in example 14copies/mL), dengue virus low concentration samples (1X 10)3copies/mL), concentration samples in Zika virus (1 extract)
104copies/mL), Zika virus low concentration samples (1X 10)3copies/mL), 10 times per sample, using ABI7500 fluorescent PCR for detection, the fluorescent reporter groups are: FAM, VIC channel, Passive Reference: NONE, reaction program as shown in table 2, calculated coefficient of variation of Ct value, and results as shown in table 3.
TABLE 3 results of repeated experiments
As can be seen from Table 3, the detection kit obtained in the embodiment 1 of the present invention performs 10 times of repeated detection on dengue virus samples and Zika virus samples with different concentrations, and the variation coefficient of the Ct value in the batch of positive samples is less than or equal to 5%, which indicates that the detection kit obtained in the embodiment 1 of the present invention has good detection repeatability and high accuracy.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
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Claims (10)
1. A primer probe combination is characterized by comprising a first primer probe group and a second primer probe group, wherein the first primer probe group comprises a dengue virus specific primer, a first Y primer and a first fluorescent probe, and the first Y primer comprises at least one of a first Y primer and a first second Y primer; the second type of primer probe group comprises Zika virus specific primers, a second type of Y primers and a second type of fluorescent probes, and the second type of Y primers comprise at least one of a second type of first Y primers and a second type of second Y primers;
wherein, the nucleotide sequence of the dengue virus specific primer is the nucleotide sequence shown in SEQ ID NO. 1-2, or the nucleotide sequence with the same function obtained by deletion, insertion or replacement of the nucleotide sequence shown in SEQ ID NO. 1-2;
the nucleotide sequence of the first Y primer is the nucleotide sequence shown in SEQ ID NO. 3, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 3;
the nucleotide sequence of the first type second Y primer is the nucleotide sequence shown in SEQ ID NO. 4, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 4;
the nucleotide sequence of the first type of fluorescent probe is the nucleotide sequence shown in SEQ ID NO. 5, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 5;
the nucleotide sequence of the Zika virus specific primer is the nucleotide sequence shown in SEQ ID NO. 6-7, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 6-7;
the nucleotide sequence of the second type first Y primer is the nucleotide sequence shown in SEQ ID NO. 8, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 8;
the nucleotide sequence of the second type second Y primer is the nucleotide sequence shown in SEQ ID NO. 9, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 9;
the nucleotide sequence of the second fluorescent probe is a nucleotide sequence shown in SEQ ID NO. 10, or a nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 10;
the fluorophore on the first type of fluorescent probe is different from the fluorophore on the second type of fluorescent probe.
2. The primer-probe combination of claim 1, wherein the first type of probe has a fluorophore FAM at the 5 'end and a quencher BHQ1 at the 3' end.
3. The primer-probe combination of claim 1, wherein the second type of probe has a fluorescent group HEX at the 5 'end and a quencher group BHQ2 at the 3' end.
4. A test kit comprising the primer-probe combination according to any one of claims 1 to 3.
5. The detection kit according to claim 4, wherein in the primer probe combination, the final concentration of the dengue virus specific primers is 0.50 μ M to 1.0 μ M, the final concentration of the first Y primer is 0.50 μ M to 1.0 μ M, and the final concentration of the first probe is 0.10 μ M to 0.30 μ M; and/or
The final concentration of the Zika virus specific primers is 0.50-1.0 mu M, the final concentration of the second type Y primers is 0.50-1.0 mu M, and the final concentration of the second type probes is 0.10-0.30 mu M.
6. The detection kit according to claim 4, further comprising at least one of a reaction buffer, an enzyme mixture, and a quality control substance.
7. The test kit of claim 6, wherein the reaction buffer comprises a one-step quantitative RT-qPCR buffer; and/or
The quality control product comprises a positive quality control product and a negative quality control product.
8. Use of the test kit according to any one of claims 4 to 7 for the detection of dengue virus and/or Zika virus.
9. Use according to claim 8, characterized in that it comprises the following steps:
providing total RNA of a sample to be detected;
performing RT-qPCR amplification on the total RNA by using the detection kit to obtain a fluorescence curve;
when the fluorescence curve is S-shaped and the Ct value is less than or equal to 38, the result is positive;
when the fluorescence curve is S-shaped and the Ct value is more than 38 and less than 40, the result is suspicious, and the total RNA is subjected to RT-qPCR amplification again;
negative results when the fluorescence curve is non-sigmoidal and Ct-value is 40 or no Ct-value.
10. The use according to claim 9, wherein the RT-qPCR amplification procedure comprises the steps of:
performing reverse transcription at 50 deg.C for 10min, and circulating for 1 time;
performing denaturation reaction at 95 deg.C for 3min, and circulating for 1 time;
amplification reaction is firstly carried out for 10s at 95 ℃, and then fluorescence is collected for 1min at 60 ℃, and the cycle frequency is 40 times.
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