CN108330210B - Zika virus, dengue virus and chikungunya virus nucleic acid detection kit and application thereof - Google Patents
Zika virus, dengue virus and chikungunya virus nucleic acid detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses a nucleic acid detection kit for Zika virus, dengue virus and chikungunya virus and application thereof, wherein the kit comprises the following components: RT-PCR reaction liquid, enzyme mixed liquid, four reaction liquids of Zika virus/dengue virus/chikungunya virus/internal standard gene, positive control and blank control. The Zika virus, dengue virus and chikungunya virus detection kit can simultaneously detect the Zika virus, dengue virus and chikungunya virus in the same reaction tube, and solves the problem that the existing in-vitro detection kit cannot simultaneously detect the Zika virus, dengue virus and chikungunya virus in the same reaction tube. In addition, the detection kit provided by the invention has the advantages of strong specificity, high sensitivity, simplicity and convenience in operation, strong repeatability, quick and objective detection result and the like, and provides an effective technical means for in vitro detection of the Zika virus, the dengue fever virus and the chikungunya virus.
Description
Technical Field
The invention relates to an in vitro diagnostic kit for mosquito-borne pathogens, in particular to a nucleic acid detection kit for Zika virus, dengue virus and chikungunya virus and application thereof, belonging to the technical field of in vitro diagnosis of viruses.
Background
Zika Virus (ZIKV) genus Flaviviridae (Flaviviridae) genus flaviviruses (flaviviruses) are spherical, about 40-70 nm in diameter, have an Envelope, have a single-stranded positive-strand RNA genome of about 10.8kb in length, include 5 'and 3' noncoding regions, and a single open reading frame in the middle, which encodes a polypeptide chain that can be cleaved to mature into 3 structural proteins and 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), and the structural proteins are capsid proteins (capsid, C), membrane precursor/membrane proteins (prememberan/membrane, prM), and Envelope proteins (Envelope, E).
Zika virus was first isolated in 1947 from the serum of monkeys in Wugandavictoria, Zika forest, and in 1948 in the same regionZIKV was also isolated from aedes africana, and thereafter, ZIKV was subsequently isolated from humans in asia and africa and various mosquito vectors, and genetic evolutionary analysis based on the NS5 gene showed that ZIKV was classified into asia and africa types. ZIKV can be cultured and propagated in cells of C6/36, Vero and the like to generate lesions, and has strong serological cross reaction with dengue virus, yellow fever virus, west nile virus and the like of the flavivirus genus. The dose of ZIKV infection causing human disease is unknown and is associated with the route of transmission. The dose for transmitting infection by mosquito bite is generally 103～105pfu. The resistance of the flavivirus virus is unknown, but the flavivirus virus is generally not acid-resistant and heat-resistant, can be inactivated at 60 ℃ for 30min, and can be inactivated by disinfectants such as 70% ethanol, 1% sodium hypochlorite, lipid solvent, peroxyacetic acid and the like and ultraviolet irradiation.
Analysis of the ZIKV genome using bioinformatics techniques showed that the virus was likely to appear between 1892-1943, approximately 1920, spread 2 times around 1940 in africa to form 2 subtypes of african type, to maleica in 1945, and to miloney in 1960 to form asia type.
In Asian regions, Malaysia, Pakistan, Cambodia, Thailand, Indonesia, isolated locally to ZIKV. In the Pacific island, Yapu island developed the first fulminant epidemic of Zika virus disease in 2007, and the strain originated in southeast Asia. The geographical location of the island of yapu is close to that of indonesia and malaysia, and although it has been reported that under the influence of wind, mosquito vectors can cross over hundreds of kilometers of ocean surface, the input of epidemic is more likely to be caused by tourism and trade activities causing people, host animals or virus-infected mosquito vectors in viremia. From 7 days in 10 months in 2013 to 6 days in 4 months in 2014, the Fassian has large-scale outbreak epidemics, the number of the outbreaks is estimated to be 32000, and meanwhile, part of virus infected persons are found to have neurological symptoms or autoimmune syndromes.
Dengue virus (Dengue virus) belongs to the flavivirus family of Flaviviridae, and is a single positive strand RNA virus divided into 4 serotypes according to the antigenicity difference of envelope protein E. The dengue fever, dengue hemorrhagic fever and dengue shock syndrome are caused mainly by the spread of Aedes aegypti and Aedes albopictus, and the incubation period is 3-14 days, usually 4-7 days after dengue virus infection. Mainly distributed in the tropics and subtropics, affecting over 100 countries and regions around the world, with 25 million people living in the threatened areas.
Chikungunya virus (CHIKV) belonging to genus alphavirus of family togaviridae, having a diameter of about 60 to 70nm and an envelope. The genome is single-strand positive-strand RNA and is about 11-12kb in length; there are 1 serotype, which can be divided into 3 genotypes, i.e. west africa, middle-east-south africa and asia. The virus can be cultured and propagated in Vero, C6/36, BHK-21, HeLa and other cells and generate lesions. The virus is not acid-resistant and heat-resistant, can be inactivated at 56 ℃ for 30 minutes, and can be killed by disinfectants such as 70% ethanol, 1% sodium hypochlorite, lipid solvent, peroxyacetic acid and the like and ultraviolet irradiation. Chikungunya Fever (CHIKF) is a viral infectious disease with Fever, joint pain/arthritis, and rash as main clinical manifestations due to infection with Chikungunya virus, and is mainly transmitted by bites of aedes albopictus and aedes aegypti. Although the fatality rate of the disease is low, the disease is easy to form large-scale outbreak and epidemic in areas with high mosquito vector density.
The real-time fluorescent PCR technology based on the fluorescent labeled probe is most widely applied to the clinical diagnosis in China at present. In the real-time fluorescent PCR reaction system of the probe method, the method can be further divided into single or multiple real-time fluorescent PCR technologies according to the number of detected pathogens, the reaction system usually comprises one or more pairs of specific PCR primers and probes, the probes and the primers are only specifically combined with corresponding templates through condition exploration in the research and development process, and the combining sites of the probes and the primers are between the two primers. The 5 'end of the probe is marked with a Reporter group (R), such as FAM, VIC, ROX and the like, and the 3' end is marked with a fluorescence quenching group (Quencher, Q), such as BHQ1, BHQ2, TAMRA and the like. When the probe is complete, the fluorescence energy emitted by the reporter group is absorbed by the quencher group, and the signal cannot be detected by the instrument. As the PCR proceeds, Taq enzyme encounters the probe bound to the template during the chain extension process, the 3 '→ 5' exonuclease activity thereof cleaves the probe, the reporter group is far away from the quencher group, and the energy thereof cannot be absorbed, i.e., a fluorescent signal is generated (FIG. 1). Therefore, the fluorescent signal has a synchronous exponential growth process as the target fragment every PCR cycle.
Most of the existing in vitro diagnostic kits aiming at the Zika virus, the dengue virus and the chikungunya virus adopt single RT-PCR reaction, if the detection of the three viruses needs to be finished, one RT-PCR reaction needs to be carried out, when large-scale sample detection is carried out, a large amount of manpower and material resources are undoubtedly consumed, and the diagnosis time is also prolonged, so that a multiple RT-PCR method which has good specificity and high sensitivity and can simultaneously detect the Zika virus, the dengue virus and the chikungunya virus is urgently needed to be established, and the method can be better and faster used for detecting the Zika virus, the dengue virus and the chikungunya virus.
Disclosure of Invention
The invention aims to solve the technical problem of providing a multiple fluorescence RT-PCR detection kit capable of respectively detecting the Zika virus, the dengue fever virus and the chikungunya virus in the same reaction tube, and the kit has the advantages of strong specificity, high sensitivity and the like.
In order to achieve the above object, the present invention adopts the following technical means:
the invention relates to a nucleic acid detection kit for Zika virus, dengue virus and chikungunya virus, which comprises the following components: RT-PCR reaction liquid, enzyme mixed liquid, four reaction liquids of Zika virus/dengue virus/chikungunya virus/internal standard gene, positive control and blank control;
wherein, the internal standard gene is a housekeeping gene;
wherein, the four reaction liquids of the Zika virus/dengue virus/chikungunya virus comprise the following 4 components:
a component (1): the kit consists of a pair of primer pairs for detecting the Zika virus and a probe for detecting the Zika virus, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.1 and SEQ ID No. 2; the base sequence of the probe is shown as SEQ ID No.3, wherein, the 5 'end of the SEQ ID No.3 is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group;
a component (2): the kit comprises a pair of primer pairs for detecting dengue viruses and a probe for detecting dengue viruses, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.4 and SEQ ID No. 5; the base sequence of the probe is shown as SEQ ID No. 6; wherein, the 5 'end of SEQ ID No.6 is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group;
a component (3): the kit comprises a pair of primer pairs for detecting the chikungunya virus and a probe for detecting the chikungunya virus, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.7 and SEQ ID No. 8; the base sequence of the probe is shown as SEQ ID No. 9; wherein, the 5 'end of SEQ ID No.9 is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group.
A component (4): the kit comprises a pair of primer pairs for detecting the housekeeping genes and a probe for detecting the housekeeping genes, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.10 and SEQ ID No. 11; the base sequence of the probe is shown as SEQ ID No. 12; wherein, the 5 'end of SEQ ID No.12 is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group;
wherein, preferably, the fluorescent reporter group is selected from FAM, VIC, ROX, CY3 or CY5 fluorescent reporter groups, and component (1), component (2), component (3) and component (4) are selected from different fluorescent reporter groups; the fluorescence quenching group is selected from any one or more of BHQ1, BHQ2, BHQ3, Dabcy1 and Tamra, but is required to be capable of quenching the fluorescence reporter group marked on the same probe.
The invention searches and optimizes the proportion of the primers and the probes, and the test results show that different proportion of the primers and the probes has obvious difference on the specificity and sensitivity of the detection results, and the invention discovers that the primers and the probes have the optimal detection effect under the following proportion through high-throughput screening tests:
in the present invention, preferably, the ratio of the primers and probes in component (1) is: the primer shown in SEQ ID No.1 is the primer shown in SEQ ID No.2, and the probe shown in SEQ ID No.3 is 400nM, 400nM and 200 nM.
In the present invention, preferably, the ratio of the primers and probes in component (2) is: the primer shown in SEQ ID No.4, the primer shown in SEQ ID No.5 and the probe shown in SEQ ID No.6 are 400nM, 400nM and 200nM, respectively.
In the present invention, preferably, the ratio of the primers and probes in component (3) is: the primer shown in SEQ ID No. 7: the primer shown in SEQ ID No.8 and the probe shown in SEQ ID No.9 are 400nM and 200nM, respectively.
In the present invention, preferably, the ratio of the primers and probes in component (4) is: the primer shown in SEQ ID No. 10: the primer shown in SEQ ID No.11 and the probe shown in SEQ ID No.12 are 200nM, 200nM and 100nM, respectively.
In the present invention, preferably, the RT-PCR reaction solution comprises: 10 XPCR buffer, 25mM Mg2+、10mM dNTPs。
In the present invention, it is preferable that the enzyme mixture solution includes: RNase inhibitors, DNA polymerases, reverse transcriptases and DNA polymerases play very important roles in PCR amplification including efficiency and specificity of reverse transcription, amplification, and more preferably, the DNA polymerase is a hot start enzyme and the reverse transcriptase is M-MLV reverse transcriptase for better effect.
In the present invention, preferably, the positive control is inactivated Zika virus/dengue virus/chikungunya virus culture solution, and the blank control is RNase-free water.
In the present invention, preferably, the detection kit is used for detecting the Zika virus, the dengue virus and the chikungunya virus according to the following steps:
(1) extracting RNA from a sample by using a nucleic acid extraction kit, wherein the sample preferably comprises: serum, plasma, mosquito vectors;
(2) the following components were prepared: 7.5ul of RT-PCR reaction solution, 5ul of enzyme mixed solution, 4ul of Zika virus/dengue virus/chikungunya virus/internal standard gene quadruple reaction solution, 3.5ul of RNase-free water and 5ul of RNA sample;
(3) and (3) RT-PCR amplification: RT-PCR amplification was performed according to the following procedure: 30min at 50 ℃; 5min at 95 ℃; at 95 ℃ for 10s and at 55 ℃ for 40s, and carrying out 45 cycles;
(4) and (4) judging a result: after the amplification is finished, judging whether to infect the corresponding virus according to the fluorescence curve;
in the invention, preferably, when the 5 'end of SEQ ID No.3 is marked with a fluorescence reporter FAM, the 3' end is marked with a fluorescence quenching group BHQ1, the 5 'end of SEQ ID No.6 is marked with a fluorescence reporter VIC, the 3' end is marked with a fluorescence quenching group BHQ1, the 5 'end of SEQ ID No.9 is marked with a fluorescence reporter ROX, the 3' end is marked with a fluorescence quenching group BHQ1, the 5 'end of SEQ ID No.12 is marked with a fluorescence reporter CY5, and the 3' end is marked with a fluorescence quenching group BHQ1, the fluorescence curve in the FAM channel is an "S" -shaped curve and the CT is less than or equal to 36.0, and the Kazaki virus is judged to be positive; judging that the Zika virus is negative if no typical S-type amplification exists or the CT is more than or equal to 36.0, and meeting the condition that a CY5 channel curve is an S-type curve and the CT is less than or equal to 35.0; the fluorescence curve in the VIC channel is an S-shaped curve, CT is less than or equal to 36.0, and the dengue fever virus is judged to be positive; no typical S-type amplification or CT more than or equal to 36.0, and the dengue fever virus is judged to be negative, and the CY5 channel curve is an S-type curve and the CT is less than or equal to 35.0; the fluorescence curve in the ROX channel is an S-shaped curve, CT is less than or equal to 36.0, and the chikungunya virus is judged to be positive; no typical S-type amplification exists or the CT is more than or equal to 36.0, the chikungunya virus is judged to be negative, and the CY5 channel curve is an S-type curve and the CT is less than or equal to 35.0.
Furthermore, the invention also provides application of the kit in preparation of reagents for detecting Zika virus, dengue virus and chikungunya virus.
Compared with the prior art, the invention has the beneficial effects that:
the detection kit for the Zika virus, the dengue virus and the chikungunya virus can be used for simultaneously detecting the Zika virus, the dengue virus and the chikungunya virus, and the problem that the existing product cannot simultaneously detect three viruses in one reaction is solved. The invention also has the advantages of simple operation, strong repeatability, quick and objective detection result and the like, and has great application prospect in the field of in vitro diagnosis in areas with high mosquito vector density.
Drawings
FIG. 1 shows a mechanism of generating a fluorescent signal of a TaqMan probe;
FIG. 2 is a graph showing the detection of mixed samples by the kit of the present invention;
FIG. 3 shows the results of a susceptibility test for detecting Zika virus using the kit of the present invention;
FIG. 4 shows the results of susceptibility test for detecting dengue virus using the kit of the present invention;
FIG. 5 shows the results of a sensitivity test for detecting chikungunya virus using the kit of the present invention;
FIG. 6 shows the results of a specificity test for detecting Zika virus using the kit of the present invention;
FIG. 7 shows the result of a specific test for detecting dengue virus using the kit of the present invention;
FIG. 8 shows the result of a specific assay for detecting chikungunya using the kit of the present invention.
Detailed Description
The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 Assembly of nucleic acid detection kits for Zika virus, dengue virus and chikungunya virus
The kit comprises the following components:
(1) RT-PCR reaction solution:
the RT-PCR reaction solution consists of RNase-free water, 10 XPCR buffer solution and 25mM Mg2+10mM dNTPs (Table 1).
TABLE 1 composition of RT-PCR reaction solution
(2) Enzyme mixture
The enzyme mixed solution comprises an RNase inhibitor, DNA polymerase, reverse transcriptase, 10 Xbuffer solution and RNase removing water. The reverse transcriptase and DNA polymerase play very important roles in PCR amplification, including efficiency and specificity of reverse transcription and amplification, and M-MLV reverse transcriptase and Taq hot start enzyme are preferably used in the present invention for better effect (Table 2).
TABLE 2 composition of enzyme mixture
| Name of reagent | Add volume (ul)/50 reaction |
| RNase inhibitors | 12.5 |
| Taq enzyme | 12.5 |
| Reverse transcriptase | 2.5 |
| 10×Buffer | 75 |
| RNase-removed water | 147.5 |
| Total of | 250 |
(3) Four-fold reaction solution of Zika virus/dengue virus/chikungunya virus/internal standard gene
The four-fold reaction liquid of the Zika virus/dengue virus/chikungunya virus comprises the following 4 components:
a component (1): the kit consists of a pair of primer pairs for detecting the Zika virus and a probe for detecting the Zika virus, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.1 and SEQ ID No. 2; the base sequence of the probe is shown as SEQ ID No.3, wherein the 5 'end of the SEQ ID No.3 is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quencher group BHQ 1;
the primer shown in SEQ ID No.1 is the primer shown in SEQ ID No.2, and the probe shown in SEQ ID No.3 is 400nM, 400nM and 200 nM.
A component (2): the kit comprises a pair of primer pairs for detecting dengue viruses and a probe for detecting dengue viruses, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.4 and SEQ ID No. 5; the base sequence of the probe is shown as SEQ ID No. 6; wherein, the 5 'end of SEQ ID No.6 is marked with a fluorescence reporter group VIC, and the 3' end is marked with a fluorescence quenching group BHQ 1;
a primer shown as SEQ ID No.4, a primer shown as SEQ ID No.5 and a probe shown as SEQ ID No.6, wherein the probe is 400nM, 400nM and 200 nM;
a component (3): the kit comprises a pair of primer pairs for detecting the chikungunya virus and a probe for detecting the chikungunya virus, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.7 and SEQ ID No. 8; the base sequence of the probe is shown as SEQ ID No. 9; wherein the 5 'end of SEQ ID No.9 is marked with a fluorescence reporter group ROX, and the 3' end is marked with a fluorescence quenching group BHQ 1.
The primer shown in SEQ ID No. 7: the primer shown in SEQ ID No.8 and the probe shown in SEQ ID No.9 are 400nM and 200nM, respectively.
A component (4): the kit comprises a pair of primer pairs for detecting the housekeeping genes and a probe for detecting the housekeeping genes, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.10 and SEQ ID No. 11; the base sequence of the probe is shown as SEQ ID No. 12; wherein the 5 'end of SEQ ID No.12 is marked with a fluorescence reporter group CY5, and the 3' end is marked with a fluorescence quenching group BHQ 1.
The primer shown in SEQ ID No. 10: the primer shown in SEQ ID No.11 and the probe shown in SEQ ID No.12 are 200nM, 200nM and 100nM, respectively.
The primer and probe sequences and the reaction liquid compositions for detecting the Zika virus, the dengue virus and the chikungunya virus are shown in the following tables 3 and 4:
TABLE 3 primers and probes for detection of Zika virus, dengue virus and chikungunya virus
TABLE 4 reaction liquid Components of Zika virus, dengue virus and chikungunya virus
| Name of reagent | Add volume (ul)/50 reaction |
| Zika virus- |
4 |
| Zika virus- |
4 |
| Zika virus-P (FAM) | 2 |
| Dengue virus- |
4 |
| Dengue virus- |
4 |
| Dengue virus-P (VIC) | 2 |
| Chikungunya virus- |
4 |
| Chikungunya virus- |
4 |
| Chikungunya virus-P (ROX) | 2 |
| Gene-F for |
2 |
| Human house gene- |
2 |
| Human housekeeping gene-P (CY5) | 1 |
(4) Positive control
Inactivated Zika virus/dengue virus/chikungunya virus culture broth (prepared from virus culture broth purchased from the center for disease prevention and control in Jiangsu province).
(5) Blank control
And (4) removing the RNase water.
Example 2 detection test of clinical samples with the kit of the invention
1. Sample processing (RNA extraction)
1.1 taking 200ul of clinical samples, adding 400ul of Binding buffered with Poly (A), fully mixing uniformly, transferring into a high-purity filter tube, centrifuging at 8000rpm for 15s, and discarding waste liquid in the collection tube.
1.2 add 500ul Inhibitor Removal Buffer to the filter tube, centrifuge at 8000rpm for 1min, discard the waste liquid in the collection tube.
1.3 Add 450ul Washing Buffer to the filter tube, centrifuge at 8000rpm for 1min, discard the waste liquid in the collection tube.
1.4 repeating the step 3, and then centrifuging at high speed for 10s, wherein the step is to remove the waste liquid completely.
1.5 adding 50ul of Elution Buffer into a filter tube, standing for 2min at room temperature, centrifuging at 8000rpm for 1min, and centrifuging to obtain a solution which is the purified RNA.
2. Reagent preparation
The preparation of the reaction system was carried out using the kit prepared in example 1 (table 5):
TABLE 5
3. PCR amplification procedure
RT-PCR amplification was performed according to the following procedure: 30min at 50 ℃; 5min at 95 ℃; at 95 ℃ for 10s and at 55 ℃ for 40s, and carrying out 45 cycles;
4. determination of results
The fluorescence curve in the FAM channel is an S-shaped curve, the CT is less than or equal to 36.0, and the Zika virus is judged to be positive; judging that the Zika virus is negative if no typical S-type amplification exists or the CT is more than or equal to 36.0, and meeting the condition that a CY5 channel curve is an S-type curve and the CT is less than or equal to 35.0; the fluorescence curve in the VIC channel is an S-shaped curve, CT is less than or equal to 36.0, and the dengue fever virus is judged to be positive; no typical S-type amplification or CT more than or equal to 36.0, and the dengue fever virus is judged to be negative, and the CY5 channel curve is an S-type curve and the CT is less than or equal to 35.0; the fluorescence curve in the ROX channel is an S-shaped curve, CT is less than or equal to 36.0, and the chikungunya virus is judged to be positive; no typical S-type amplification exists or the CT is more than or equal to 36.0, the chikungunya virus is judged to be negative, and the CY5 channel curve is an S-type curve and the CT is less than or equal to 35.0.
The specific results of the 5 clinical samples are shown in the following table 6, and the detection results of the mixed samples are shown in fig. 2:
TABLE 65 test results of clinical specimens
Test example 1 results of sensitivity test of the kit of the present invention
Extracted nucleic acids of a standard reference strain of Zika virus, dengue virus and a standard reference strain of chikungunya virus (prepared from a virus culture solution purchased from the center for disease prevention and control in Jiangsu province) were diluted 10-fold. The experimental results showed that the kit of the present invention (prepared in example 1) could detect 0.001PFU and that the CT value was changed in a gradient with decreasing concentration (fig. 3, fig. 4, fig. 5). The test result shows that the kit has high sensitivity for diagnosing Zika virus, dengue virus and chikungunya virus.
Test example 2 test results of specificity of the kit of the present invention
In order to determine the specificity of the kit for detecting Zika virus, dengue virus and chikungunya virus of the present invention, Zika virus, dengue virus and chikungunya virus nucleic acid detection kit prepared in example 1 was used to detect Zika virus, dengue virus and chikungunya virus, and an internal standard template was added. The results showed that the FAM channel amplified only zika virus (fig. 6); VIC channel only amplified dengue virus (fig. 7); the ROX channel amplifies chikungunya virus only (fig. 8); CY5 is an internal standard signal derived from a human housekeeping gene; and the channels do not cross react with each other.
Sequence listing
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atatcggcag tgcgccagca agg 23
<210> 10
<211> 24
<212> DNA
<213> Artificial sequence
<400> 10
atggcggaga tctttctaaa taag 24
<210> 11
<211> 24
<212> DNA
<213> Artificial sequence
<400> 11
ccgcgtttca ctctctatat ttct 24
<210> 12
<211> 27
<212> DNA
<213> Artificial sequence
<400> 12
tctgtaaagt gccacagaga cgagctc 27
Claims (11)
1. A nucleic acid detection kit for Zika virus, dengue virus and chikungunya virus is characterized by comprising the following components: the kit comprises RT-PCR reaction liquid, enzyme mixed liquid, four reaction liquids of Zika virus/dengue virus/chikungunya virus/internal standard gene, positive control and blank control, wherein the internal standard gene is a human housekeeping gene;
wherein, the quadruple reaction solution comprises the following 4 components:
a component (1): the kit consists of a pair of primer pairs for detecting the Zika virus and a probe for detecting the Zika virus, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.1 and SEQ ID No. 2; the base sequence of the probe is shown as SEQ ID No.3, wherein, the 5 'end of the SEQ ID No.3 is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group;
a component (2): the kit comprises a pair of primer pairs for detecting dengue viruses and a probe for detecting dengue viruses, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.4 and SEQ ID No. 5; the base sequence of the probe is shown as SEQ ID No. 6; wherein, the 5 'end of SEQ ID No.6 is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group;
a component (3): the kit comprises a pair of primer pairs for detecting the chikungunya virus and a probe for detecting the chikungunya virus, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.7 and SEQ ID No. 8; the base sequence of the probe is shown as SEQ ID No. 9; wherein, the 5 'end of SEQ ID No.9 is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group;
a component (4): the kit comprises a pair of primer pairs for detecting the housekeeping genes and a probe for detecting the housekeeping genes, wherein the base sequences of the primer pairs are respectively shown as SEQ ID No.10 and SEQ ID No. 11; the base sequence of the probe is shown as SEQ ID No. 12; wherein, the 5 'end of SEQ ID No.12 is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group;
component (1), component (2), component (3) and component (4) are selected from different fluorescent reporter groups.
2. The test kit according to claim 1, characterized in that: the fluorescence reporter group is selected from FAM, VIC, ROX or CY5 fluorescence reporter group, and the fluorescence quenching group is selected from BHQ 1.
3. The test kit according to claim 1, characterized in that: the proportion of the primers and the probes in the component (1) is as follows: the primer shown in SEQ ID No.1 is the primer shown in SEQ ID No.2, and the probe shown in SEQ ID No.3 is =400nM, 400nM and 200 nM.
4. The test kit according to claim 1, characterized in that: the proportion of the primers and the probes in the component (2) is as follows: primer shown as SEQ ID No.4 and probe shown as SEQ ID No.6 =400nM and 200nM, respectively.
5. The test kit according to claim 1, characterized in that: the proportion of the primers and the probes in the component (3) is as follows: the primer shown in SEQ ID No. 7: primer shown in SEQ ID No.8 and probe =400nM 200nM shown in SEQ ID No. 9.
6. The test kit according to claim 1, characterized in that: the proportion of the primers and the probes in the component (4) is as follows: the primer shown in SEQ ID No. 10: primer shown in SEQ ID No.11 probe shown in SEQ ID No.12 = 200nM:100 nM.
7. The detection kit according to claim 1, wherein the RT-PCR reaction solution comprises: 10 XPCR buffer, 25mM Mg2+、10mM dNTPs。
8. The test kit according to claim 1, wherein the enzyme mixture comprises: rnase inhibitor, DNA polymerase, reverse transcriptase, said DNA polymerase being a hot start enzyme and said reverse transcriptase being M-MLV reverse transcriptase.
9. The test kit according to claim 1, characterized in that: the positive control is inactivated Zika virus/dengue virus/chikungunya virus culture solution, and the blank control is RNA enzyme-removed water.
10. The test kit according to any one of claims 1 to 9, characterized in that: when the detection kit is used for detecting Zika virus, dengue fever virus and chikungunya virus, the detection kit is carried out according to the following steps:
(1) extracting RNA of a sample by adopting a nucleic acid extraction kit;
(2) the following components were prepared: 7.5 mul of RT-PCR reaction solution, 5 mul of enzyme mixed solution, 4 mul of Zika virus/dengue virus/chikungunya virus/internal standard gene quadruple reaction solution, 3.5 mul of RNase removing water and 5 mul of RNA sample;
(3) and (3) RT-PCR amplification: RT-PCR amplification was performed according to the following procedure: 30min at 50 ℃; 5min at 95 ℃; at 95 ℃ for 10s and at 55 ℃ for 40s, and carrying out 45 cycles;
(4) and (4) judging a result: after the amplification is finished, judging whether to infect the corresponding virus according to the fluorescence curve;
when the 5 'end of SEQ ID No.3 is marked with a fluorescence reporter FAM, the 3' end is marked with a fluorescence quenching group BHQ1, the 5 'end of SEQ ID No.6 is marked with a fluorescence reporter VIC, the 3' end is marked with a fluorescence quenching group BHQ1, the 5 'end of SEQ ID No.9 is marked with a fluorescence reporter ROX, the 3' end is marked with a fluorescence quenching group BHQ1, the 5 'end of SEQ ID No.12 is marked with a fluorescence reporter CY5, and the 3' end is marked with a fluorescence quenching group BHQ1, the fluorescence curve in the FAM channel is an S-shaped curve, the CT is less than or equal to 36.0, and the Zika virus is judged to be positive; judging that the Zika virus is negative if no typical S-type amplification exists or the CT is more than or equal to 36.0, and meeting the condition that a CY5 channel curve is an S-type curve and the CT is less than or equal to 35.0; the fluorescence curve in the VIC channel is an S-shaped curve, CT is less than or equal to 36.0, and the dengue fever virus is judged to be positive; no typical S-type amplification or CT more than or equal to 36.0, and the dengue fever virus is judged to be negative, and the CY5 channel curve is an S-type curve and the CT is less than or equal to 35.0; the fluorescence curve in the ROX channel is an S-shaped curve, CT is less than or equal to 36.0, and the chikungunya virus is judged to be positive; no typical S-type amplification exists or the CT is more than or equal to 36.0, the chikungunya virus is judged to be negative, and the CY5 channel curve is an S-type curve and the CT is less than or equal to 35.0.
11. Use of a kit according to any one of claims 1 to 10 for the preparation of reagents for the detection of Zika virus, dengue virus and chikungunya virus.
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