CN106755571B

CN106755571B - Kit for detecting Zika virus, dengue fever virus and chikungunya virus

Info

Publication number: CN106755571B
Application number: CN201611080920.4A
Authority: CN
Inventors: 张岩; 盖伟; 单万水; 邢婉丽; 宋翠丹; 马桂红; 刘厚明; 程京
Original assignee: Boao Biological Group Co ltd; Third Peoples Hospital of Shenzhen
Current assignee: Boao Biological Group Co ltd; Third Peoples Hospital of Shenzhen
Priority date: 2016-11-30
Filing date: 2016-11-30
Publication date: 2020-08-07
Anticipated expiration: 2036-11-30
Also published as: CN106755571A

Abstract

The invention discloses a kit for detecting Zika virus, dengue fever virus and chikungunya virus. The invention provides a protective primer probe combination, which consists of a primer probe group I, a primer probe group II and a primer probe group III; the primer probe group I consists of a primer ZIKV-F, a primer ZIKV-R and a probe ZIKV-P; the primer probe group II consists of a primer DENV-F, a primer DENV-R1, a primer DENV-R2 and a probe DENV-P; the primer probe group III consists of a primer CHIKV-F, a primer CHIKV-R and a probe CHIKV-P; the primer ZIKV-F, the primer ZIKV-R, the probe ZIKV-P, the primer DENV-F, the primer DENV-R1, the primer DENV-R2, the probe DENV-P, the primer CHIKV-F, the primer CHIKV-R and the probe CHIKV-P are sequentially shown as sequences 1 to 10. The invention has great value for the detection of Zika virus, dengue fever virus and chikungunya fever virus and the prevention and control of related diseases.

Description

Kit for detecting Zika virus, dengue fever virus and chikungunya virus

Technical Field

The invention relates to a kit for detecting Zika virus, dengue virus and chikungunya virus.

Background

Mosquito vector viruses are widely distributed all over the world, are important infectious disease pathogens, are mainly transmitted by mosquito bites, have high transmission speed, are easy to cause serious harm to human beings and the society, and particularly, in recent years, the epidemic situation of the mosquito vector viruses is continuously developed. Most of the mosquito-borne viruses belong to the genus yellow fever virus of the family yellow fever virus, mainly including yellow fever virus, dengue virus and Zika virus, and a few of the genus alphaviruses of the family membranous virus, such as chikungunya virus, all of which belong to mosquito-borne viruses, and in recent years, epidemic outbreaks occur continuously. The epidemic situation of Zika virus outbreaks in Brazil in 2015-2016 is rapidly spread to more than 30 countries and regions, meanwhile, input cases are reported in Europe, North America and other countries, and many input cases are also reported in China this year. In recent years, dengue and chikungunya viruses have been the mosquito-borne viruses responsible for the outbreaks of the largest-scale infectious disease, and the world health organization has reported about 50 ten thousand cases of dengue each year.

At present, national research institutions develop a Zika virus detection method, a dengue virus detection method and a chikungunya virus detection method based on a real-time fluorescent quantitative PCR probe method, but the methods are single-index virus detection methods. If the related mosquito vector viruses need to be screened at the same time, a plurality of kits are needed, the sample consumption is large, the operation is complex, and the cost is high.

At present, because DNA polymerase and reverse transcriptase in a reagent must be stored at the temperature of minus 20 ℃, the nucleic acid detection reagent based on a real-time fluorescent quantitative PCR probe method needs dry ice to ensure cold chain transportation and low-temperature storage and ensure that a kit does not lose efficacy. However, since mosquito-associated viruses such as Zika virus, dengue virus, chikungunya virus, etc. are frequently concentrated in tropical regions such as Africa, south America and southeast Asia, and are limited by local climatic conditions and economic conditions, it is very difficult to realize cold-chain transportation of reagents in most of the regions, so that the application of these nucleic acid detection reagents in these regions is greatly limited.

Disclosure of Invention

The invention aims to provide a kit for detecting Zika virus, dengue virus and chikungunya virus.

The method comprises the following steps of firstly protecting a primer probe combination (primer probe combination A), wherein the primer probe combination comprises a primer probe group I, a primer probe group II and a primer probe group III;

the primer probe group I consists of a primer ZIKV-F, a primer ZIKV-R and a probe ZIKV-P;

the primer ZIKV-F is (b1) or (b 2);

(b1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;

(b2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;

the primer ZIKV-R is (b3) or (b 4);

(b3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

(b4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;

the nucleotide sequence of the probe ZIKV-P is as follows (b5) or (b 6);

(b5) a single-stranded DNA molecule shown as sequence 3 in the sequence table;

(b6) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;

the primer probe set II consists of a primer DENV-F, a primer DENV-R1, a primer DENV-R2 and a probe DENV-P;

the primer DENV-F is (c1) or (c 2);

(c1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

(c2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;

the primer DENV-R1 is (c3) or (c 4);

(c3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;

(c4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and having the same functions as the sequence 5;

the primer DENV-R2 is (c5) or (c 6);

(c5) a single-stranded DNA molecule shown in sequence 6 of the sequence table;

(c6) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 6 and having the same functions as the sequence 6;

the nucleotide sequence of the probe DENV-P is as follows (c7) or (c 8);

(c7) a single-stranded DNA molecule shown in sequence 7 of the sequence table;

(c8) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 7 and having the same functions as the sequence 7;

the primer probe group III consists of a primer CHIKV-F, a primer CHIKV-R and a probe CHIKV-P;

the primer CHIKV-F is (d1) or (d 2);

(d1) a single-stranded DNA molecule shown in sequence 8 of the sequence table;

(d2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 8 and have the same functions as the sequence 8;

the primer CHIKV-R is (d3) or (d 4);

(d3) a single-stranded DNA molecule shown in sequence 9 of the sequence table;

(d4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 9 and has the same function as the sequence 9;

the nucleotide sequence of the probe CHIKV-P is as follows (d5) or (d 6);

(d5) a single-stranded DNA molecule shown in sequence 10 of the sequence table;

(d6) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 10 and has the same function as the sequence 15.

The tail end of the probe ZIKV-P is marked with a fluorophore A, the tail end of the probe DENV-P is marked with a fluorophore B, and the tail end of the probe CHIKV-P is marked with a fluorophore C. The fluorescent group A, the fluorescent group B and the fluorescent group C are different fluorescent groups.

Specifically, the probe ZIKV-P has a fluorescent group JOE at the 5 'end and a quencher group BHQ2 at the 3' end.

Specifically, the probe DENV-P has a fluorescent group FAM at the 5 'end and a quencher group BHQ1 at the 3' end.

Specifically, probe CHIKV-P has a fluorescent group CY5 at the 5 'end and a quencher group BHQ3 at the 3' end.

The application of the primer probe combination A is as follows (e1) or (e 2):

(e1) identifying a Zika virus and/or a dengue virus and/or a chikungunya virus;

(e2) and detecting whether the sample to be detected contains Zika virus and/or dengue virus and/or chikungunya virus.

The invention also provides a kit (kit A) comprising the primer probe combination A.

The kit A comprises a reaction tube C, a primer ZIKV-F, a primer ZIKV-R, a probe ZIKV-P, a primer DENV-F, a primer DENV-R1, a primer DENV-R2, a probe DENV-P, a primer CHIKV-F, a primer CHIKV-R and a probe CHIKV-P are embedded in the reaction tube C, the primer and the probe are specifically embedded in the reaction tube by means of an encapsulating reagent, the encapsulating reagent is composed of 3-10 parts by mass of agarose, 1-5 parts by mass of saponin and 2-6 parts by mass of hydroxypropyl cellulose, the encapsulating reagent is specifically composed of 5 parts by mass of agarose, 3 parts by mass of saponin and 4 parts by mass of hydroxypropyl cellulose sodium, the reaction tube is specifically an eight-row PCR reaction tube, the preparation method of the reaction tube C is specifically characterized in that the encapsulating reagent, the primer ZIKV-F, the primer ZIKV-R, the probe ZIKV-P, the primer CHINV-R, the probe and the probe are mixed solution with the concentration of the probe ZIKV-2 mu, the probe concentration of the probe embedded liquid is 387-2 mu, the concentration of the dry solution of the embedded primer, the probe 2 mu 3-2 mu-2-mu-V-embedded in the reaction tube, and the probe concentration of the dry-embedded primer, and the probe concentration of the probe-2-V-embedded primer, and the probe concentration of the probe-3-V-embedded.

The kit A also comprises a reaction component A and/or a reaction component B.

Reaction component a (colorless transparent liquid): consists of solute and solvent; the solvent is Tris-HCl buffer solution with the pH value of 8.0-8.4 and the concentration of 50 mM; solutes and their concentrations in reaction component a are as follows: KCl 40-100mM, MgCl₂1.5-4mM, dNTP 0.2-0.4mM (i.e. concentrations of dATP, dTTP, dCTP and dGTP are all 0.2-0.4mM), hot start Taq DNA polymerase 0.1-0.5U/. mu.l, RNase inhibitor 0.5U/. mu.l.

The reaction component B (in a dry powder state) is AMV reverse transcriptase.

The reaction component A can be specifically: consists of solute and solvent; the solvent is Tris-HCl buffer solution with the pH value of 8.2 and the concentration of 50 mM; solutes and their concentrations in reaction component a are as follows: KCl 70mM, MgCl₂2.8mM, dNTP 0.3mM (i.e., concentrations of dATP, dTTP, dCTP and dGTP are all 0.3mM), hot start Taq DNA polymerase 0.3U/. mu.l, RNase inhibitor 0.5U/. mu.l.

The kit A also comprises a positive quality control substance and/or a negative quality control substance.

Positive quality control product: comprises a Zika virus RNA standard (single-stranded RNA molecule, the nucleic acid sequence of which is shown in sequence 11 of the sequence table) and a dengue virus RNA standard (single-stranded RNA molecule, the nucleic acid of which is shown in sequence 11 of the sequence table)The sequence is shown as sequence 12 in the sequence table) and a chikungunya virus RNA standard substance (a single-stranded RNA molecule, the nucleic acid sequence of which is shown as sequence 13 in the sequence table). In the positive quality control product, the nucleic acid concentration of each virus can be 10⁴copies/μl。

Negative quality control product: inactivated clinical negative samples without Zika virus, dengue virus and chikungunya virus.

The kit A is used as the following (e1) or (e 2):

(e1) identifying a Zika virus and/or a dengue virus and/or a chikungunya virus;

The invention also protects the application of the kit A, which is (e1) or (e 2):

(e1) identifying a Zika virus and/or a dengue virus and/or a chikungunya virus;

The method for detecting whether the virus to be detected is Zika virus, dengue fever virus or chikungunya virus by using the kit A comprises the following steps: adding nucleic acid of the virus to be detected, the reaction component A and the reaction component B into the reaction tube C, and then detecting; if the detection channel of the fluorophore A shows positive, the virus to be detected is or is selected as Zika virus; if the detection channel of the fluorophore B shows positive, the virus to be detected is or is selected as dengue virus; if the detection channel of the fluorophore C shows positive, the virus to be detected is or is selected as chikungunya virus.

The method for detecting whether the virus to be detected is Zika virus, dengue fever virus or chikungunya virus by using the kit A comprises the following specific steps:

(1) taking 50U of the reaction component B, adding 100 mul of 50% glycerol, standing until the solution is transparent, uniformly mixing by vortex, and performing instantaneous centrifugation;

(2) taking reaction tubes C, and adding 10. mu.l of the reaction component A, 1. mu.l of the solution obtained in step (1) and 2-9. mu.l to each tubeAdding ddH into the solution to be tested₂O to the total volume of 20 μ l, then the cover of the reaction tube was closed, vortexed, mixed, and centrifuged instantaneously, followed by detection (the Zika virus detection channel was JOE, the dengue virus detection channel was FAM, and the chikungunya virus detection channel was CY 5);

for the JOE detection channel, the detection result is Zika virus positive for the sample with the Ct value less than or equal to 37.5, and the detection result is Zika virus negative for the sample with the Ct value greater than 37.5; for the sample with the Ct value less than or equal to 37 of the FAM detection channel, the detection result is positive for the dengue virus, and for the sample with the Ct value more than 37 of the FAM detection channel, the detection result is negative for the dengue virus; and for the sample with the CY5 detection channel Ct value less than or equal to 36.7, the detection result is positive by the chikungunya virus, and for the sample with the CY5 detection channel Ct value more than 36.7, the detection result is negative by the chikungunya virus.

The method for detecting whether the sample to be detected contains Zika virus and/or dengue virus and/or chikungunya virus by using the kit A is as follows: adding nucleic acid of the virus to be detected, the reaction component A and the reaction component B into the reaction tube C, and then detecting; if the detection channel of the fluorophore A shows positive, the sample to be detected contains or is suspected to contain the Zika virus; if the detection channel of the fluorescent group B shows positive, the sample to be detected contains or is suspected to contain dengue fever virus; and if the detection channel of the fluorophore C shows positive, the sample to be detected contains or is suspected to contain the chikungunya virus.

The method for detecting whether the sample to be detected contains Zika virus and/or dengue virus and/or chikungunya virus by using the kit A is as follows:

(2) taking a reaction tube C, adding 10 mu l of the reaction component A, 1 mu l of the solution obtained in the step (1) and 2-9 mu l of the solution to be detected into each tube, and adding ddH₂O to a total volume of 20. mu.l, then the reaction tube was capped, vortexed, centrifuged instantaneously, and then assayed (the Zika virus assay channel was JOE, dengue virus assayChannel set to FAM, chikungunya virus detection channel set to CY 5);

The program settings for any of the above described tests are as follows: 15-30min at 50 ℃; 3-5min at 95 ℃; 5-15s at 95 ℃ and 30-60s at 60 ℃ for 45 cycles. The program setting of any one of the above detection is specifically as follows: 23min at 50 ℃; 4min at 95 ℃; 95 ℃ for 10s, 60 ℃ for 45s, 45 cycles.

The invention also provides a primer probe combination (primer probe combination B) which is (a2) or (a3) as follows:

(a2) any two of the primer probe group I, the primer probe group II and the primer probe group III are used;

(a3) the primer probe group I, the primer probe group II or the primer probe group III.

The invention also protects the application of the primer probe combination B, which is (e1) or (e 2):

(e1) identifying a Zika virus and/or a dengue virus and/or a chikungunya virus;

The invention also protects a primer combination which is (f1), (f2) or (f 3):

(f1) the primer group I, the primer group II and the primer group III are combined;

(f2) any two of the primer group I, the primer group II and the primer group III are used;

(f3) the primer group I, the primer group II or the primer group III.

The invention also protects the application of the primer combination, which is (e1) or (e 2):

(e1) identifying a Zika virus and/or a dengue virus and/or a chikungunya virus;

The invention also provides a method for detecting whether the virus to be detected is Zika virus, dengue fever virus or chikungunya virus, which comprises the following steps: detecting nucleic acid of the virus to be detected; if the nucleic acid contains the specific fragment M1 and the virus to be detected is or is candidate to be Zika virus; if the nucleic acid contains the specific fragment M2-1 or M2-2, the virus to be detected is dengue virus or candidate; if the nucleic acid contains the specific fragment M3, the virus to be detected is or is selected as chikungunya virus.

The invention also provides a method for detecting whether the sample to be detected contains Zika virus and/or dengue virus and/or chikungunya virus, which comprises the following steps: detecting nucleic acid of a sample to be detected; if the nucleic acid contains the specific fragment M1 and the sample to be tested contains or is suspected to contain the Zika virus; if the nucleic acid contains the specific fragment M2-1 or M2-2, the sample to be tested contains or is suspected to contain dengue virus; if the nucleic acid contains the specific fragment M3, the sample to be tested contains or is suspected to contain the chikungunya virus.

The specific fragment M1 is (f1), (f2) or (f3) as follows:

(f1) target sequences of the primer ZIKV-F and the primer ZIKV-R in Zika virus nucleic acid;

(f2) DNA molecule shown as 59 th-159 th nucleotide of sequence 11 in the sequence table;

(f3) a DNA molecule shown in a sequence 11 of a sequence table.

The specific fragment M2-1 is (g1), (g2) or (g 3):

(g1) target sequences of said primer DENV-F and said primer DENV-R1 in a dengue virus (dengue virus type I, dengue virus type II, or dengue virus type III) nucleic acid;

(g2) a DNA molecule shown as 912 nd and 1033 rd nucleotides of a sequence 12 in a sequence table;

(g3) a DNA molecule shown as a sequence 12 in a sequence table.

The specific fragment M2-2 is (g4), (g5) or (g 6):

(g4) target sequences of said primer DENV-F and said primer DENV-R2 in dengue virus (dengue virus type IV) nucleic acid;

(g5) DNA molecule shown as nucleotide 942-1083 of sequence 14 in the sequence table;

(g6) a DNA molecule shown as a sequence 14 in a sequence table.

The specific fragment M3 is (h1), (h2) or (h3) as follows:

(h1) target sequences of the primer CHIKV-F and the primer CHIKV-R in Zika virus nucleic acid;

(h2) a DNA molecule shown as 874-961 th nucleotide of the sequence 13 in the sequence table;

(h3) a DNA molecule shown in a sequence 13 of a sequence table.

Any of the dengue virus type II strains described above may in particular be the DEN-2NGC strain.

The kit provided by the invention has the following advantages: (1) the Zika virus, the dengue virus and the chikungunya virus can be detected in the same reaction system; (2) has good sensitivity (the Zika virus, the dengue virus and the chikungunya virus can detect 50 copies/reaction nucleic acid samples, and the Zika virus detection sensitivity can even reach 20 copies/reaction); (3) has good specificity; (4) at present, because enzymes are unstable at normal temperature, most of related nucleic acid detection reagents need cold chain transportation, a large amount of dry ice is used in the cold chain transportation process, the dry ice is extremely expensive, and the transportation cost is improved. The invention has great value for the detection of Zika virus, dengue fever virus and chikungunya fever virus and the prevention and control of related diseases.

Drawings

FIG. 1 shows the results corresponding to RNA1 in example 2.

FIG. 2 shows the results corresponding to RNA2-1 in example 2.

FIG. 3 shows the results corresponding to RNA3 in example 2.

FIG. 4 shows the results of example 3.

FIG. 5 shows that in example 4, the RNA concentration was 10²Average Ct value of copies/. mu.l of test solution.

FIG. 6 shows that in example 4, the RNA concentration was 10¹Average Ct value of copies/. mu.l of test solution.

Detailed Description

The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention, the experimental procedures in the following examples are conventional, unless otherwise specified, the test materials used in the following examples are commercially available from conventional biochemical reagent stores, quantitative tests in the following examples are provided in triplicate, and the results are averaged, RNase inhibitor Promega corporation, Cat No. N2611. AMV reverse transcriptase (dry powder state), L ife sciences advanced Technologies, Cat No. L RT 20116,63452U/mg agarose Baygene corporation, Cat No. 162090.

Ginsenoside: sigma company, cat #: 65839.

the chemical structural formula of the ginsenoside is shown in formula (I).

Hydroxypropyl cellulose (average M)_w100000): sigma Co, cat # 191884.

The chemical structural formula of the hydroxypropyl cellulose is shown in a formula (II).

Example 1 preparation of kit

Preparation of primers and probes

A primer probe group A for detecting Zika Virus (Zika Virus) consists of a primer ZIKV-F, a primer ZIKV-R and a probe ZIKV-P. The primer ZIKV-F is shown as a sequence 1 in a sequence table. The primer ZIKV-R is shown as a sequence 2 in a sequence table. The nucleotide sequence of the probe ZIKV-P is shown as a sequence 3 in a sequence table, the 5 'end of the probe ZIKV-P is provided with a fluorescent group JOE, and the 3' end of the probe ZIKV-P is provided with a quenching group BHQ 2.

ZIKV-F：5'-CAGCTGGCATCATGAAGAAYC-3'；

ZIKV-R：5'-CACYTGTCCCATCTTYTTCTCC-3'；

ZIKV-P：5'-JOE-CYGTTGTGGATGGAATAGTGG-BHQ2-3'。

Primer probe set B for detecting Dengue virus (Dengue virus) consists of primer DENV-F, primer DENV-R1, primer DENV-R2 and probe DENV-P. The primer DENV-F is shown as a sequence 4 in a sequence table. The primer DENV-R1 is shown as a sequence 5 in a sequence table. The primer DENV-R2 is shown as a sequence 6 in the sequence table. The nucleotide sequence of the probe DENV-P is shown as a sequence 7 in a sequence table, the 5 'end of the probe DENV-P is provided with a fluorescent group FAM, and the 3' end of the probe DENV-P is provided with a quenching group BHQ 1.

DENV-F：5'-GACTAGAGGTTAGAGGAGACCCCC-3'；

DENV-R1：5'-CCATTCCATTTTCTGGCGTTCTG-3'；

DENV-R2：5'-CAATCCATCTTGCGGCGCTCT-3'；

DENV-P：5'-FAM-CTGTCTCYWCAGCATCATTCCAGGCA-BHQ1-3'。

The primer probe group C for detecting Chikungunya virus (Chikungunya virus) consists of a primer CHIKV-F, a primer CHIKV-R and a probe CHIKV-P. The primer CHIKV-F is shown as a sequence 8 in a sequence table. The primer CHIKV-R is shown as a sequence 9 in a sequence table. The nucleotide sequence of the probe CHIKV-P is shown as a sequence 10 in a sequence table, the 5 'terminal of the probe CHIKV-P is provided with a fluorescent group CY5, and the 3' terminal of the probe CHIKV-P is provided with a quenching group BHQ 3.

CHIKV-F：5'-AAAGGGCAAACTYAGCTTCAC-3'；

CHIKV-R：5'-GCCTGGGCTCATCGTTATTC-3'；

CHIKV-P：5'-CY5-CGCTGTGATACAGTGGTTTCGTGTG-BHQ3-3'。

In the nucleotide sequences of the above respective primers and probes, Y represents C or T, and W represents A or T.

Preparation of kit

The kit consists of the following components:

reaction component a (colorless transparent liquid): consists of solute and solvent; the solvent is Tris-HCl buffer solution with the pH value of 8.2 and the concentration of 50 mM; solutes and their concentrations in reaction component a are as follows: KCl 70mM, MgCl₂2.8mM, dNTP 0.3mM (i.e., concentrations of dATP, dTTP, dCTP and dGTP are all 0.3mM), hot start Taq DNA polymerase 0.3U/. mu.l, RNase inhibitor 0.5U/. mu.l.

Reaction component B (dry powder state): AMV reverse transcriptase.

The reaction tube C is an eight-connected calandria tube, a primer ZIKV-F, a primer ZIKV-R, a probe ZIKV-P, a primer DENV-F, a primer DENV-R1, a primer DENV-R2, a probe DENV-P, a primer CHIKV-F, a primer CHIKV-R and a probe CHIKV-P are embedded at the bottom of each tube, ① a packaging reagent, the primer ZIKV-F, the primer ZIKV-R, the probe ZIKV-P, the primer DENV-F, the primer DENV-R1, the primer DENV-R2, the probe DENV-P, the primer ZIKV-F, the primer CHIKV-R, the probe CHIKV-P and sterile water are mixed to obtain an embedding liquid (the concentration of the packaging reagent is 0.1g/100ml, the concentration of the primer ZIKV-F is 2 mu M, the concentration of the primer ZIKV-R is 2 mu M, the concentration of the CHIKV-R is 1 mu M, the concentration of the CHIKV-R is 2 mu M, the concentration of the primer DENV-R is 2 mu M, the primer is 2 mu M, the dry sodium carbonate, the concentration of the primer DENV-R is 387-P, and the 5 mu M concentration of the 5 mu M of the dry sodium is added to react in the tube.

Positive quality control product: a nucleic acid solution containing a Zika virus RNA standard (a single-stranded RNA molecule, the nucleic acid sequence of which is shown in sequence 11 of the sequence table), a dengue virus RNA standard (a single-stranded RNA molecule, the nucleic acid sequence of which is shown in sequence 12 of the sequence table) and a chikungunya virus RNA standard (a single-stranded RNA molecule, the nucleic acid sequence of which is shown in sequence 13 of the sequence table), wherein the nucleic acid concentration of each virus is 10⁴copies/μl。

Third, using method of kit

The solution to be detected can be the solution I or the solution II. Solution I: taking a virus to be detected, extracting nucleic acid (extracting genome DNA if a sample to be detected is a DNA virus, and extracting total RNA if the sample to be detected is an RNA virus), and obtaining a nucleic acid solution or a diluent thereof, namely the solution I. Solution II: taking a sample to be detected (such as serum or plasma), extracting total RNA, and obtaining an RNA solution or a diluent thereof as a solution II.

1. Taking 50U of reaction component B, adding 100 mul of 50% glycerol, standing for a few minutes until the solution becomes transparent, vortex and uniformly mixing for 15 seconds, centrifuging instantaneously, and storing at-20 ℃. 50% of glycerin: glycerol and water were mixed in equal volumes to give a solution.

2. Taking reaction tubes C, adding 10 mu l of reaction component A, 1 mu l of solution obtained in step 1 and 2-9 mu l of solution to be detected into each tube, and adding ddH₂O to the total volume of 20 mu l, then the cover of the reaction tube is covered, the mixture is intermittently vortexed and mixed for 30s, and the mixture is instantaneously centrifuged and then detected.

Setting a detection channel: the detection channel of the Zika virus is JOE, the detection channel of the dengue virus is FAM, and the detection channel of the chikungunya virus is CY5, and three fluorescence channels are simultaneously detected during detection.

The program settings were as follows: 23min at 50 ℃; 4min at 95 ℃; 95 ℃ for 10s, 60 ℃ for 45s, 45 cycles.

And (4) judging the standard: for the JOE detection channel, the detection result is Zika virus positive for the sample with the Ct value less than or equal to 37.5, and the detection result is Zika virus negative for the sample with the Ct value greater than 37.5; for the sample with the Ct value less than or equal to 37 of the FAM detection channel, the detection result is positive for the dengue virus, and for the sample with the Ct value more than 37 of the FAM detection channel, the detection result is negative for the dengue virus; and for the sample with the CY5 detection channel Ct value less than or equal to 36.7, the detection result is positive by the chikungunya virus, and for the sample with the CY5 detection channel Ct value more than 36.7, the detection result is negative by the chikungunya virus.

Fourthly, the advantages of the kit

The kit provided by the invention completes the reverse transcription process and the polymerase chain reaction process in one reaction system, and can effectively avoid the pollution in the preparation process of a PCR amplification system after the reverse transcription is performed to prepare the cDNA in a two-step method.

Example 2 sensitivity and specificity analysis for three mosquito-borne Virus nucleic acid detection kits

Firstly, preparation of template

A Zika virus RNA standard (single-stranded RNA molecule, the nucleic acid sequence of which is shown in SEQ ID NO: 11 in the sequence Listing) was prepared as RNA 1.

A dengue virus RNA standard (single-stranded RNA molecule, the nucleic acid sequence of which is shown in sequence 12 of the sequence listing) was prepared as RNA 2-1. RNA2-1 corresponds to dengue virus types I, II and III.

A dengue virus RNA standard (single-stranded RNA molecule, the nucleic acid sequence of which is shown in sequence 14 of the sequence listing) was prepared as RNA 2-2. RNA2-2 corresponds to dengue virus type IV.

A chikungunya virus RNA standard (single-stranded RNA molecule, the nucleic acid sequence of which is shown in sequence 13 of the sequence table) was prepared as RNA 3.

Taking RNA (RNA1 or RNA2-1 or RNA2-2 or RNA3), diluting with sterile water to obtain RNA concentration of 10¹⁰copies/. mu.l RNA solution. Taking RNA solution, and performing gradient dilution with sterile water to respectively obtain RNA concentration of 10⁴copies/μl、10³copies/μl、10²copies/μl、10¹copies/. mu.l, 4 copies/. mu.l dilutions.

Secondly, sensitivity and cross specificity analysis of the kit

The kit prepared in the second step of example 1 was used to perform the assay according to the third step of example 1, and the solution to be assayed was the diluent prepared in the first step, and the amount added was 5. mu.l.

The results for RNA1 are shown in FIG. 1 and Table 1. In FIG. 1, each two similar curves are shown as a set, representing two replicates, and the curves from left to right correspond to dilutions with RNA concentration varying from high to low. For the Zika virus RNA standard, the RNA concentration of the test solution was 10⁴copies/μl、10³copies/μl、10²copies/μl、10¹The copy virus detection channels show obvious S-shaped amplification curves, and the Ct value is less than 37.5, and the result is positive. For the Zika virus RNA standard, the RNA concentration of the test solution was 10⁴copies/μl、10³copies/μl、10²copies/μl、10¹No S-shaped amplification curve is shown on the dengue virus detection channel at copies/mu l or 4 copies/mu l, no Ct value exists, and the result is negative. For the Zika virus RNA standard, the RNA concentration of the test solution was 10⁴copies/μl、10³copies/μl、10²copies/μl、10¹The copies/mu l or 4 copies/mu l, the chikungunya virus detection channel does not show S-shaped amplification curve, no Ct value exists, and the result is negative. For negative quality control products, the Zika virus detection channel, the dengue virus detection channel and the chikungunya virus detection channel do not display S-shaped amplification curves, Ct values are absent, and the amplification results are negative.

TABLE 1

The results for RNA2-1 are shown in FIG. 2 and Table 2. In FIG. 2, each two similar curves are shown as a set, representing two replicates, and the curves from left to right correspond to dilutions with increasing RNA concentration. For dengue virus RNA standard, the RNA concentration of the test solution is 10⁴copies/μl、10³copies/μl、10²copies/μl、10¹The copy virus detection channels do not show S-shaped amplification curves, have no Ct value and have negative results. For dengue virus RNA standard, the RNA concentration of the test solution is 10⁴copies/μl、10³copies/μl、10²copies/. mu.l or 10¹The copies/mu l and dengue virus detection channels all show obvious sigmoid amplification curves, and the Ct value is less than 37, and the result is positive. For dengue virus RNA standard, the RNA concentration of the test solution is 10⁴copies/μl、10³copies/μl、10²copies/μl、10¹The copies/mu l or 4 copies/mu l, the chikungunya virus detection channel does not show S-shaped amplification curve, no Ct value exists, and the result is negative. For negative quality control products, the Zika virus detection channel, the dengue virus detection channel and the chikungunya virus detection channel do not display S-shaped amplification curves, Ct values are absent, and the amplification results are negative.

TABLE 2

The corresponding results for RNA2-2 are consistent with those for RNA 2-1.

The results for RNA3 are shown in FIG. 3 and Table 3. In FIG. 3, each two similar curves are shown as a set, representing two replicates, and the curves from left to right correspond to dilutions with increasing RNA concentration. For the chikungunya virus RNA standard, the RNA concentration of the test solution is 10⁴copies/μl、10³copies/μl、10²copies/μl、10¹The copy virus detection channels do not show S-shaped amplification curves, have no Ct value and have negative results. For the chikungunya virus RNA standard, the RNA concentration of the test solution is 10⁴copies/μl、10³copies/μl、10²copies/μl、10¹No S-shaped amplification curve is shown on the dengue virus detection channel at copies/mu l or 4 copies/mu l, no Ct value exists, and the result is negative. For the chikungunya virus RNA standard, the RNA concentration of the test solution is 10⁴copies/μl、10³copies/μl、10²copies/. mu.l or 10¹The copies/mu l and the chikungunya virus detection channel all show obvious S-shaped amplification curves, the Ct value is less than 36.7, and the result is positive. For negative quality control products, the Zika virus detection channel, the dengue virus detection channel and the chikungunya virus detection channel do not display S-shaped amplification curves, Ct values are absent, and the amplification results are negative.

TABLE 3

Example 3 specificity analysis of the kit

Common pathogens with similar symptoms to three mosquito-borne virus infections serve as specific references.

The samples to be tested were as follows:

hepatitis b virus: zhangjuan, Chenjianzong, Zhangjin Ping, Jiaxin, Jianjiang mountain; the action of astragaloside A in vitro against hepatitis B virus; fourth department of military medical university, 2008,28(24): 2291-.

Epstein barr virus (human herpesvirus type 4): wufang, Dianthus hainanensis, Geliu willow, Qiyanwei, Gaohui; 245 test cases of human herpesviruses types 1-4 in saliva of human immunodeficiency virus infected persons; huaxi journal of oral medicine 2012,30(5):514 and 517.

Measles virus L4, Zhao Dapeng, Yao, Wu Xiao Juan, Zi Juan spring, Wangchun, determination of the complete sequence of the genome of measles virus L4, national Biometrics conference, 2007.

H5N1 subtype avian influenza virus A/duck/Guangdong/S1322/2010 (DK/GD/S1322/2010): rukunpeng, sideroc fly, zhangfang, wangjingjing, xiaohong; preparing and identifying an HA protein monoclonal antibody of H5 subtype avian influenza virus; 453 in 2015(5) 457 in veterinary science of China.

Subtype H7N2 Avian influenza virus (Avian in flu virus, AIV) A/Chicken/Hebei/1/2002: wangxiang, Tianke Cong, Wang Wei, Sun Ming, Ying Xiu Ling; separating and identifying the H _7N _2 subtype avian influenza virus CK/HB/1/02 strain in China; chinese virology 2005,20(6):632 and 636.

Group A rotavirus TB-Chen strain; well known, plum-plectrum, fang yan chun, gao, wei billows; preliminary study of the genotypes and the NSP5/NSP6 of the TB-Chen strain rotavirus; 349 and 354 in the journal of viral academic, 2009 (5).

Human cytomegalovirus: wangming, Maojianping; human cytomegalovirus detection techniques; 103-107 in journal of biological engineering of China 2007,27 (2).

Zika virus: zhanghuo, Lidexin; zika virus and Zika virus; virus school newspaper, 2016 month 1, vol.32, stage 1, 121- & 123.

Dengue virus type ii (DEN-2NGC strain): shuliman, Leili, Limoni, Haomai, Chenabing; detecting the NS1 gene of dengue fever virus and enzyme digestion typing thereof by using a universal primer; as a journal of the Guiyang medical college, 2004,29(4): 283-286.

Chikungunya virus: wangyu, zhuan yubo, wangyai, zhao Guiliang; progress in diagnostic techniques for chikungunya virus infection; 522 and 524 in J.Clin.TEST, 2012 and 7.

The kit prepared in step two of example 1 was used. And (3) extracting nucleic acid of each sample to be detected (extracting genome DNA if the sample to be detected is DNA virus, and extracting total RNA if the sample to be detected is RNA virus) to serve as a solution to be detected, and detecting according to the method of the third step in the example 1, wherein the adding amount of the solution to be detected is 5 mu l.

The partial detection results are shown in FIG. 4.

For positive quality control products, the Zika virus detection channel, the dengue virus detection channel and the chikungunya virus detection channel are all positive results. For negative quality control products, the Zika virus detection channel, the dengue virus detection channel and the chikungunya virus detection channel are all negative results.

For Zika virus: the Zika virus detection channel shows an obvious S-shaped amplification curve, and the Ct value is less than 37.5, and the result is positive; the dengue virus detection channel does not display an S-shaped amplification curve, has no Ct value and has a negative result; the chikungunya virus detection channel does not show an S-shaped amplification curve, has no Ct value, and has a negative result.

For dengue virus type ii: the Zika virus detection channel does not display an S-shaped amplification curve, has no Ct value and has a negative result; the dengue virus detection channel shows an obvious sigmoid amplification curve, and the Ct value is less than 37, and the result is positive; the chikungunya virus detection channel does not show an S-shaped amplification curve, has no Ct value, and has a negative result.

For chikungunya virus: the Zika virus detection channel does not display an S-shaped amplification curve, has no Ct value and has a negative result; the dengue virus detection channel does not display an S-shaped amplification curve, has no Ct value and has a negative result; the chikungunya virus detection channel shows an obvious S-shaped amplification curve, the Ct value is less than 36.7, and the result is positive.

All other viruses, the Zika virus detection channel, the dengue virus detection channel and the chikungunya virus detection channel, were negative results.

The result shows that the kit provided by the invention has good specificity for the amplification of three mosquito-borne viruses.

Example 4 analysis of nucleic acid detection kit for three mosquito-borne viruses by simulating transportation stability at Normal temperature

The kit prepared in step two of example 1 was used.

And placing the reaction component A, the reaction component B and the reaction tube C on a shaking table, simulating a normal-temperature transportation process by oscillating at room temperature, and detecting the sample to be detected as a component of the kit after 0 day, 3 days, 5 days, 10 days, 15 days, 20 days and 30 days respectively.

The samples to be tested are respectively as follows: dilution of Zika virus RNA standard (Single-stranded RNA molecule, the nucleic acid sequence of which is shown in sequence 11 of the sequence table) with RNA concentration of 10²copies/. mu.l or 10¹copies/. mu.l; diluting dengue virus RNA standard (single-stranded RNA molecule, the nucleic acid sequence of which is shown as sequence 12 in the sequence table) with RNA concentration of 10²copies/. mu.l or 10¹copies/. mu.l; diluting chikungunya virus RNA standard (single-stranded RNA molecule, the nucleic acid sequence of the single-stranded RNA molecule is shown as sequence 13 in a sequence table) with the RNA concentration of 10²copies/. mu.l or 10¹copies/μl。

The test was carried out in the same manner as in step three of example 1, except that the amount of the solution to be tested was 5. mu.l.

Each solution to be tested was subjected to 8 replicates and the Ct values averaged.

RNA concentration of 10²The average Ct value of copies/. mu.l of test solution is shown in FIG. 5. RNA concentration of 10¹The average Ct value of copies/. mu.l of test solution is shown in FIG. 6. The activity of the reagent can be maintained for at least 30 days under the normal-temperature transportation condition, the detection sensitivity is unchanged, and the problem of normal-temperature transportation in areas such as south America and the like can be solved.

Example 5 detection of clinical samples

The solution to be tested is a serum sample of a suspected mosquito-borne virus infected patient (141 cases in total), and the serum sample is provided by an Ecuador disease control center.

Each serum sample was divided into two portions, one portion was assayed by the kit prepared in step two of example 1 in accordance with the method of step three of example 1, and the amount of the solution to be assayed was 5. mu.l, and the other portion was assayed by the commercially available E L ISA kit.

The results are shown in Table 4.

TABLE 4

In addition, for a few samples, the detection result of the commercially available E L ISA kit is negative, but the detection result of the kit is positive, and virus culture verification is carried out on the samples, wherein the detection result of the kit is positive by adopting the commercially available E6332 ISA kit.

Sequence listing

<120> a kit for detecting Zika virus, dengue virus and chikungunya virus

<130>GNCYX161840

<160>14

<210>1

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(20)

<223>y = c or t

<400>1

cagctggcat catgaagaay c 21

<210>2

<211>22

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(4,16)

<223>y = c or t

<400>2

cacytgtccc atcttyttct cc 22

<210>3

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(2)

<223>y = c or t

<400>3

cygttgtgga tggaatagtg g 21

<210>4

<211>24

<212>DNA

<213> Artificial sequence

<400>4

gactagaggt tagaggagac cccc 24

<210>5

<211>23

<212>DNA

<213> Artificial sequence

<400>5

ccattccatt ttctggcgtt ctg 23

<210>6

<211>21

<212>DNA

<213> Artificial sequence

<400>6

caatccatct tgcggcgctc t 21

<210>7

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(8)

<223>y = c or t

<220>

<221>misc_feature

<222>(9)

<223>w = a or t

<400>7

ctgtctcywc agcatcattc caggca 26

<210>8

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(13)

<223>y = c or t

<400>8

aaagggcaaa ctyagcttca c 21

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<400>9

gcctgggctc atcgttattc 20

<210>10

<211>25

<212>DNA

<213> Artificial sequence

<400>10

cgctgtgata cagtggtttc gtgtg 25

<210>11

<211>990

<212>RNA

<213> Artificial sequence

<400>11

auguacuuga ucccaggccu acaagcggca gcagcgcgug cugcccagaa aaggacagca 60

gcuggcauca ugaagaaucc cguuguggau ggaauagugg uaacugacau ugacacaaug 120

acaauagacc cccaggugga gaagaagaug ggacaagugu uacucauagc aguagccauc 180

uccagugcug ugcugcugcg gaccgccugg ggaugggggg aggcuggagc ucugaucaca 240

gcagcgaccu ccaccuugug ggaaggcucu ccaaacaaau acuggaacuc cucuacagcc 300

accucacugu gcaacaucuu cagaggaagc uaucuggcag gagcuucccu uaucuauaca 360

gugacgagaa acgcuggccu gguuaagaga cguggaggug ggacgggaga gacucuggga 420

gagaagugga aagcucgucu gaaucagaug ucggcccugg aguucuacuc uuauaaaaag 480

ucagguauca cugaagugug uagagaggag gcucgccgug cccucaagga uggaguggcc 540

acaggaggac augccguauc ccggggaagu gcaaagauca gaugguugga ggagagagga 600

uaucugcagc ccuaugggaa gguuguugac cucggaugug gcagaggggg cuggagcuau 660

uaugccgcca ccauccgcaa agugcaggag gugagaggau acacaaaggg aggucccggu 720

caugaagaac ccaugcuggu gcaaagcuau ggguggaaca uaguucgucu caagagugga 780

guggacgucu uccacauggc ggcugagccg ugugacacuc ugcuguguga cauaggugag 840

ucaucaucua guccugaagu ggaagagaca cgaacacuca gagugcucuc uauggugggg 900

gacuggcuug aaaaaagacc aggggccuuc uguauaaagg ugcugugccc auacaccagc 960

acuaugaugg aaaccaugga gcgacugcaa 990

<210>12

<211>1055

<212>RNA

<213> Artificial sequence

<400>12

aacaagugcc uuuuuguuca caccacuucc accagcugau caugaaggau gggagggaaa 60

uaguggugcc augccgcaac caagaugaac uuguggguag ggcuagagua ucacaaggug 120

cuggguggag ccugagagaa accgcaugcc uaggcaaauc auaugcacaa auguggcagc 180

ugauguacuu ccacaggaga gaccugagac uagccgcuaa ugccaucugu ucagccguuc 240

caguugauug ggucccaacc agccgcacca ccuggucgau ccaugcccau caccaaugga 300

ugacaacaga agacaugcug ucagugugga auaggguuug gauagaggaa aacccaugga 360

uggaggacaa aacccauaua uccaauuggg aagauguucc auauuuaggg aaaagggaag 420

aucaauggug uggaucccug auaggcuuaa cagcaagggc caccugggcc accaacauac 480

aaguggccau aaaccaagug agaagacuaa uugggaauga gaauuaucug gauuacauga 540

caucaaugaa gagauucaag aacgagagug aucccgaagg ggcacucugg ugagucaaca 600

cauuuauaaa auaaaggaaa acaagaaauu aaacaaggca agaagucagg ccggauuaag 660

ccauaguacg guaagagcua ugcugccugu gagccccguc uaaggacgua aaaugaaguc 720

aggccgaaag ccacgguuug agcaaaccgu gcugccugua gcuccaucgu ggggauguaa 780

aaaccuggga ggcugcaacc cauggaagcu guacgcaugg gguagcagac uagugguuag 840

aggagacccc ucccgaaaca uaacgcagca gcggggccca acaccagggg aagcuguacc 900

cuggugguaa ggacuagagg uuagaggaga ccccccgcau aacaauaaac agcauauuga 960

cgcugggaga gaccagagau ccugcugucu cuacagcauc auuccaggca cagaacgcca 1020

gaaaauggaa uggugcuguu gaaucaacag guucu 1055

<210>13

<211>990

<212>RNA

<213> Artificial sequence

<400>13

auggcugcgu gagacacacg uagccuacca guuucuuacu gcucuacucu gcaaagcaag 60

agauuaagaa cccaucaugg auccugugua cguggacaua gacgcugaca gcgccuuuuu 120

gaaggcccug caacgugcgu accccauguu ugagguggaa ccuaggcagg ucacaccgaa 180

ugaccaugcu aaugcuagag cguucucgca ucuagcuaua aaacuaauag agcaggaaau 240

ugaucccgac ucaaccaucc uggauauugg uagugcgcca gcaaggagga ugaugucgga 300

caggaaguac cacugcguuu gcccgaugcg cagugcagaa gaucccgaga gacucgccaa 360

uuaugcgaga aagcuagcau cugccgcagg aaaaguccug gacagaaaca ucucuggaaa 420

gaucggggac uuacaagcag uaauggccgu gccagacacg gagacgccaa cauucugcuu 480

acacacagau guaucaugua gacagagagc agacgucgcg auauaccaag acgucuaugc 540

uguacacgca cccacgucgc uauaccacca ggcgauuaaa gggguccgau uggcguacug 600

gguaggguuu gacacaaccc cguucaugua caaugccaug gcgggugccu accccucaua 660

cucgacaaau ugggcagaug agcagguacu gaaggcuaag aacauaggau uauguucaac 720

agaccugacg gaagguagac gaggcaaauu gucuauuaug agaggaaaaa agcuagaacc 780

gugcgaccgu gugcuguucu caguaggguc aacgcucuac ccggaaagcc guaagcuacu 840

uaagagcugg caccuaccau cgguguucca uuuaaagggc aagcucagcu ucacaugccg 900

cugugauaca gugguuucgu gcgaaggcua cgucguuaag agaauaacga ugagcccagg 960

ccuuuacgga aaaaccacag gguaugcggu 990

<210>14

<211>1083

<212>RNA

<213> Artificial sequence

<400>14

cccuuagaug agagguuuag caccucccuc cucuucuuga augacauggg aaaggugagg 60

aaagauaucc cgcaguggga accauccaag ggauggaaaa acuggcaaga gguuccuuuu 120

ugcucccacc auuuccacaa gaucuucaug aaagauggcc gcucacuagu uguuccaugc 180

agaaaccagg augaacugau aggaagagcc agaaucucgc agggggcugg auggagcuug 240

agagagacag ccugucuggg caaagcuuac gcccaaaugu ggucgcuuau guauuuccau 300

agaagggacc ugcgccuagc cuccauggcc auaugcucag caguuccaac agaaugguuu 360

ccaacaagca gaacaacaug gucaauccac gcucaucacc aguggaugac cacugaagau 420

augcuuaaag uguggaacag aguguggaua gaagauaacc ccaauaugac ugacaagacu 480

ccaguccauu cgugggaaga cauaccuuac cuagggaaaa gagaggaucu guggugcgga 540

ucccugauug gacuuucuuc cagagccacc ugggcgaaga auauucacac ggcuauaacc 600

caggucagga aucugaucgg aaaagaggaa uauguggauu acaugccagc caugaaaaga 660

uacagcgcuc cuuucgagag ugaaggaguu cuguaauugu uaacaacaaa caccaaaggg 720

accauugaag ucaggccacu ugugccacgg cuugagcaaa ccgugcugcc uguagcuccg 780

ccaauaaugg gaggcguaaa auucccaggg aggccaugcg ccacggaagc uguacgcgug 840

gcauauugga cuagcgguua gaggagaccc cucccaucac ugacaaaacg cagcaaaaaa 900

gggggcccga agccaggagg aagcuguacu ccugguggaa ggacuagagg uuagaggaga 960

cccccccaac acaaaaacag cauauugacg cugggaaaga ccagagaucc ugcugucucu 1020

gcaacaucaa uccaggcaca gagcgccgca agauggauug guguuguuga uccaacaggu 1080

ucu 1083

Claims

1. The primer probe combination consists of a primer probe group I, a primer probe group II and a primer probe group III;

the primer probe group I consists of a primer ZIKV-F, a primer ZIKV-R and a probe ZIKV-P; the primer ZIKV-F is a single-stranded DNA molecule shown in a sequence 1 in a sequence table; the primer ZIKV-R is a single-stranded DNA molecule shown in a sequence 2 of a sequence table; the nucleotide sequence of the probe ZIKV-P is a single-stranded DNA molecule shown as a sequence 3 in a sequence table;

the primer probe set II consists of a primer DENV-F, a primer DENV-R1, a primer DENV-R2 and a probe DENV-P; the primer DENV-F is a single-stranded DNA molecule shown in a sequence 4 in a sequence table; the primer DENV-R1 is a single-stranded DNA molecule shown in a sequence 5 in a sequence table; the primer DENV-R2 is a single-stranded DNA molecule shown in a sequence 6 in a sequence table; the nucleotide sequence of the probe DENV-P is a single-stranded DNA molecule shown as a sequence 7 in a sequence table;

the primer probe group III consists of a primer CHIKV-F, a primer CHIKV-R and a probe CHIKV-P; the primer CHIKV-F is a single-stranded DNA molecule shown in a sequence 8 of a sequence table; the primer CHIKV-R is a single-stranded DNA molecule shown in a sequence 9 of a sequence table; the nucleotide sequence of the probe CHIKV-P is a single-stranded DNA molecule shown as a sequence 10 in a sequence table;

the tail end of the probe ZIKV-P is marked with a fluorophore A, the tail end of the probe DENV-P is marked with a fluorophore B, and the tail end of the probe CHIKV-P is marked with a fluorophore C; the fluorescent group A, the fluorescent group B and the fluorescent group C are different fluorescent groups.

2. A kit comprising the primer probe combination of claim 1.

3. A kit comprising a reaction tube C; the reaction tube C is embedded with the primer ZIKV-F, the primer ZIKV-R, the probe ZIKV-P, the primer DENV-F, the primer DENV-R1, the primer DENV-R2, the probe DENV-P, the primer CHIKV-F, the primer CHIKV-R and the probe CHIKV-P in claim 1.

4. The kit of claim 2 or 3, wherein: the kit also comprises a reaction component A and/or a reaction component B;

the reaction component A: consists of solute and solvent; the solvent is Tris-HCl buffer solution with the pH value of 8.0-8.4 and the concentration of 50 mM; solutes and their concentrations in reaction component a are as follows: KCl 40-100mM, MgCl₂1.5-4mM, 0.2-0.4mM dNTP, 0.1-0.5U/mul hot start Taq DNA polymerase and 0.5U/mul RNase inhibitor;

the reaction component B: AMV reverse transcriptase.

5. The primer combination consists of a primer group I, a primer group II and a primer group III;

the primer group I consists of a primer ZIKV-F and a primer ZIKV-R in claim 1;

the primer group II consists of the primer DENV-F, the primer DENV-R1 and the primer DENV-R2 in the claim 1;

the primer set III consists of the primer CHIKV-F and the primer CHIKV-R in claim 1.

6. Use of a primer combination according to claim 5 for the preparation of a kit; the use of the kit is as follows (e1) or (e 2):

(e1) identifying a Zika virus and/or a dengue virus and/or a chikungunya virus;