CN108531647A - A kind of zika virus one-step method fluorescence rt-PCR detection methods and kit - Google Patents

Abstract

The invention provides a Zika virus one-step fluorescent rt-PCR detection method and a kit. The present invention designs a pair of specific primers and a probe for detecting the genetic marker, and its nucleotide sequences are respectively shown in SEQ ID NO.1-3. The present invention also provides the application of the specific primer pair and/or probe, a kit for detecting Zika virus, and a one-step fluorescent rt-PCR detection method for Zika virus. The fluorescence quantitative PCR reaction of the present invention can be carried out continuously in the same reaction tube, the operation is simple, and the pollution can be effectively prevented. The detection method is fast, accurate and sensitive, and can eliminate the influence of cross-reaction of Zika virus and other flaviviruses on serological detection, and has good specificity; it also has broad-spectrum and wide clinical adaptability, with broadly application foreground.

Description

A kind of Zika virus one-step fluorescent rt-PCR detection method and kit

technical field

The invention belongs to the field of biological detection, in particular to a one-step fluorescent rt-PCR detection method and kit for Zika virus.

Background technique

Zika virus (ZIKV) is a mosquito-borne flavivirus first isolated from febrile rhesus monkeys in Uganda in 1947 and is related to other globally relevant arthropod-borne human pathogens, including dengue fever (DENV), yellow Fever virus (YFV), West Nile (WNV), Japanese encephalitis (JEV) and tick-borne encephalitis virus. In the past decade, ZIKV infection has caused pandemics in Micronesia, French Polynesia, and South and Central America. In most cases, ZIKV infection results in a self-limited febrile illness associated with rash and conjunctivitis, but can also lead to severe neurologic disorders including Guillain-Barré syndrome and meningoencephalitis. Infection in pregnant women is of particular concern because of its association with severe fetal abnormalities, including microcephaly, spontaneous abortion, and intrauterine growth restriction with placental insufficiency.

The virus is a full-length unsegmented single-stranded positive-sense RNA(+) virus that contains two flanking non-coding regions, 5'NCR and 3'NCR. The 5' end of the genome has a typical methylated cap structure for cellular translation, and the 3' end is non-polyadenylated to form a circular structure. Virus particle RNA is infectious and simultaneously functions as genomic RNA and viral messenger RNA, co-translated and post-translationally processed into multimeric proteins by host and viral proteases. The current laboratory diagnostic methods for Zika virus include etiological methods, serological methods, and detection techniques for viral nucleic acids.

The traditional etiological method mainly uses cell culture to isolate the virus, which is time-consuming and labor-intensive. Moreover, the toxemia of Zika virus lasts for a short time, making it difficult to collect suitable samples. Serological detection methods, including ELISA, immunofluorescence, neutralization test, etc., are also widely used in laboratory detection of Zika virus. However, virus-specific IgM and neutralizing antibodies appeared late, and could not be detected until about one week after the onset of disease. At the same time, cross-reactivity between flaviviruses was common and difficult to identify.

Contents of the invention

The first purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a genetic marker for detecting Zika virus.

The second object of the present invention is to provide specific primer pairs and probes for detecting Zika virus.

The third object of the present invention is to provide the application of the specific primer pair and/or probe.

The fourth object of the present invention is to provide a kit for detecting Zika virus.

The fifth object of the present invention is to provide a one-step fluorescent rt-PCR detection method for Zika virus based on the specific primer pair and/or probe.

The object of the present invention is achieved through the following technical solutions:

A genetic marker for detecting Zika virus, the nucleotide sequence of which is shown in SEQ ID NO.4. In addition, those skilled in the art should understand that the specific fragment of this sequence can also be used as a genetic marker for detecting Zika virus.

Application of the genetic marker for detecting Zika virus in the preparation of a kit for detecting Zika virus.

According to the conserved sequence, the present invention designs specific primer pairs and probes for detecting the genetic marker through repeated comparison and screening. The specific primer pair for detecting Zika virus described is:

The upstream primer sequence is: 5'-GGTTCTCATCAATGGTTTTGCT-3'; (as shown in SEQ ID NO.1)

The downstream primer sequence is: 5'-TGGAACAAACCATCGCTCGTA-3'. (as shown in SEQ ID NO.2)

The fluorescent probe used in conjunction with the specific primer pair has a sequence of 5'-TGGCCTGGTTGGCA-3' (as shown in SEQ ID NO.3), and a fluorescent reporter group is labeled at the 5' end of the sequence , labeled with a fluorescent quencher at its 3' end.

The fluorescent reporting group can be at least one of FAM, TET, VIC, HEX, ROX, TAMRA, CY3, CY3.5, CY5 and CY5.5; the fluorescent quenching group can be MGB and At least one of the BHQs.

Application of the specific primer pair and/or probe in the preparation of a kit for detecting Zika virus.

A kit for detecting Zika virus, containing the pair of specific primers.

The kit for detecting Zika virus also includes the fluorescent probe.

The kit for detecting Zika virus may also include at least one of positive standard, negative quality control, RT-PCR reaction solution, reverse transcriptase, PCR enzyme, and fluorescent signal calibration reagent.

The positive standard product is a carrier plasmid inserted with the genetic marker for detecting Zika virus.

The negative quality control substance can be sterile DEPC water.

The RT-PCR reaction solution is composed of four nucleotides, dATP, dUTP, dGTP and dCTP, and a buffer solution containing magnesium ions.

The fluorescence signal correction reagent, such as ROX Reference Dye or Dye II (50×), can be used to correct the fluorescence signal error between wells.

A Zika virus one-step fluorescent rt-PCR detection method, utilizing the specific primer pair shown in SEQ ID NO.1, SEQ ID NO.2 and the fluorescent probe shown in SEQ ID NO.3 to carry out real-time fluorescence quantification PCR, to detect Zika virus in the sample to be tested.

The optimal reaction conditions of the real-time fluorescent quantitative PCR are: reverse transcription at 42°C for 5 minutes, 95°C, pre-denaturation for 10s; denaturation at 95°C for 5s, fluorescence detection at 60°C for 34s, 40 cycles.

In the PCR reaction system, the final concentration of the primer is preferably 0.1-1 μM, more preferably 0.2 μM; the final concentration of the fluorescent probe is preferably 0.1-0.5 μM, more preferably 0.3 μM.

The Ct value of the sample amplification curve <34 is positive for Zika virus; the sample Ct value >34 is negative for Zika virus, and the copy number of Zika virus can be calculated for positive samples according to the standard curve.

Compared with the prior art, the present invention has the following advantages and effects:

(1) This invention establishes a fast, accurate and sensitive fluorescent quantitative PCR detection method, the extraction of viral RNA can be completed within 0.5 hours, qPCR can be realized within 1.5 hours, and the entire detection method takes less than 2 hours, further improved Good support for my country's Zika virus prevention and control work.

(2) The present invention can eliminate the influence of cross-reaction of Zika virus and other flaviviruses on serological detection, and has good specificity.

(3) The one-step fluorescent quantitative PCR reaction of the present invention can be continuously carried out in the same reaction tube, the operation is simple, and pollution can be effectively prevented.

(4) The fluorescent quantitative PCR Zika detection method established by the present invention has a broad spectrum, covering two subtypes of Zika virus Asian strains and African strains; it can also be used for the detection of samples such as clinical serum, and has wide adaptability.

(5) The one-step fluorescent rt-PCR detection method provided by the present invention can detect a minimum copy number of Zika virus of 20 copies/mL, which has very good sensitivity.

Description of drawings

Figure 1 is an analysis diagram of the dye method amplification curve results of the primers in Example 1; wherein, the abscissa cycle represents the number of cycles, and the ordinate represents the fluorescence intensity.

Fig. 2 is the result analysis diagram of the dye method melting curve of the primer in embodiment 1; Wherein, abscissa Temperature represents the melting temperature Tm value of PCR reaction, and ordinate is the second-order negative derivative of fluorescence intensity.

Figure 3 is the result analysis diagram of the amplification curve of the plasmid standard product in Example 2; in the figure, S1～S7 respectively represent the concentrations of 2.0×10 ⁷ copies/mL, 2.0×10 ⁶ copies/mL, and 2.0×10 ⁵ copies/mL , 2.0×10 ⁴ copies/mL, 2.0×10 ³ copies/mL, 2.0×10 ² copies/mL, 2.0×10 ¹ copies/mL plasmid standard amplification curves, the abscissa cycle represents the number of cycles, and the ordinate Indicates the fluorescence intensity.

Fig. 4 is the result analysis chart of the standard curve of the plasmid standard product in Example 2; wherein, the abscissa represents the logarithmic value of the plasmid copy concentration, and the ordinate Ct represents the fluorescence threshold.

Fig. 5 is the result analysis diagram of the amplification curve detection Zika virus Asian type in embodiment 4; Wherein, A is the detection sample of Zika virus Asian type strain, B is the positive quality control product, the abscissa cycle represents the number of cycles, and the ordinate represents The fluorescence intensity.

Fig. 6 is the analysis diagram of the results of the Zika virus African-type amplification curve detected in Example 5; wherein, A is the Zika virus African-type strain detection sample, B is a positive quality control product, the abscissa cycle represents the number of cycles, and the ordinate represents The fluorescence intensity.

7 is an analysis diagram of the amplification curve results of the detection of Zika virus patient serum in Example 6; wherein, A is a test sample of a patient, B is a test sample of another patient, and C is a positive quality control.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

The selection and specific primer and probe design of embodiment 1 Zika virus conserved sequence

Zika virus strains with complete genome sequences were obtained from the NCBI database, strains with close homology were removed, regionally representative Zika virus strains were selected, and the conserved sequences were obtained by comparison using the Clustal W program. Select a segment with no secondary structure and a high degree of conservation (its nucleotide sequence is shown in SEQ ID NO.4), and use the Primer express design software to design multiple pairs of primers and probes. The length of the primers is generally about 20 bases. The 5' end of the probe is designed with a fluorescent reporter group FAM, and the 3' end group is MGB. In this experiment, multiple pairs of primers and probes were designed, and the optimal primer and probe sequence combinations were finally screened as follows:

Upstream primer: GGTTCTCATCAATGGTTTTGCT; (SEQ ID NO.1)

Downstream primer: TGGAACAACCATCGCTCGTA; (SEQ ID NO.2)

Probe: FAM-TGGCCTGGTTGGCA-MGB. (SEQ ID NO.3)

Zika virus (KU955589.1, provided by Guangzhou Provincial Center for Disease Control and Prevention) cell culture fluid was used to detect the specificity of the optimal primers of the present invention.

1. Extraction of viral RNA

According to the reagent manufacturer's instructions, TRIZOL (purchased from Takara, product number 9109) nucleic acid extraction method was used to extract the ZIKV virus nucleic acid in the Zika virus (KU955589.1) cell culture medium.

Take an appropriate amount of virus-containing tissue or cultured cells, add an appropriate amount of RNAiso Plus and homogenize, let stand at room temperature for 5 minutes, centrifuge at 12,000g, 4°C for 5 minutes, and transfer the supernatant to a new 1.5mL centrifuge tube. Add 1/5 volume of RNAiso Plus in chloroform, vortex to mix, let stand at room temperature for 5 minutes, and centrifuge at 12,000g for 15 minutes at 4°C. Transfer the supernatant to a new centrifuge tube, add 0.5 to 1 times the volume of RNAiso Plus isopropanol, let stand at room temperature for 10 minutes, and centrifuge at 12,000 g at 4°C for 10 minutes. Wash the pellet with 75% ethanol equal to that of RNAiso Plus, and centrifuge at 7,500g°C for 5 minutes. Discard the supernatant to keep the precipitate, dry (do not heat and dry), dissolve in an appropriate amount of DEPC-treated water, and set aside.

2. Reverse transcription of cDNA

The extracted viral RNA was reverse-transcribed into cDNA by random primer PCR (purchased from Takara, RR047A).

(1) Genomic DNA removal reaction

Prepare the reaction mixture on ice according to the following components. In order to ensure the accuracy of the preparation of the reaction solution, when performing each reaction, prepare the Master Mix according to the number of reactions + 2, and then distribute it into each reaction tube. Add the RNA sample last.

Table 1 Removal of Genomic DNA Reaction System

Remarks: ^*1 : In a 20μL reverse transcription reaction system, the SYBR Green qPCR method can use up to 1μg of viral RNA.

(2) Reverse transcription reaction

The reaction solution was prepared on ice. In order to ensure the accuracy of the preparation of the reaction solution, when performing various reactions, the Master Mix should be prepared according to the number of reactions + 2, and then 10 μL should be dispensed into each reaction tube*3. Immediately proceed to the reverse transcription reaction after gentle mixing.

Table 2 Reverse transcription reaction system

The reaction conditions are as follows: reverse transcription at 37°C for 15min ^*3 , denaturation at 85°C for 5sec, and storage at 4°C. *4

Remark:

*2: The reverse transcription system can be expanded accordingly.

*3: When there is non-specific amplification in the PCR reaction, raising the temperature to 50°C will improve it.

*4: When the synthesized cDNA needs to be stored for a long time, please store it at -20°C or lower.

(3) Primer specific detection

The reverse-transcribed cDNA of Zika virus RNA was used as a template, and the improved TaKaRa Ex Taq HS (Takara, RR820A) was used for PCR amplification. The specificity of the optimal primer was monitored by detecting the fluorescence intensity of SYBR Green I in the reaction solution. sex.

Prepare the PCR reaction solution according to the following components (please prepare the reaction solution on ice). To account for pipetting errors, the volume of the prepared master mix should be at least 10% greater than the total volume for all reactions.

Table 3 reaction system

*1: The final concentration of the primer is preferably 0.1 to 1 μM. (Figure 1 and Figure 2 are the results of the final primer concentration of 0.2 μM)

*2: In a 25μL reaction system, the amount of DNA template added is usually below 100ng. If necessary, serial dilution can be performed to determine the optimal amount of DNA template added.

On-machine detection: the selected fluorescent quantitative PCR instrument (model is Bio-Rad CFX 96 real-time fluorescent quantitative PCR instrument), the reaction conditions are as follows:

Step 1: 95°C for 30 seconds;

Step 2: PCR reaction GOTO: 39 (40 cycles) 95°C for 5 seconds;

60°C for 30 seconds

Step 3: Melt Curve

qPCR (SYBR Green I) primer debugging results show that the primer pair of the present invention has a good amplification curve and can amplify Zika virus fragments (see Figure 1). It can be seen from FIG. 2 that the melting curve of the primers has a single peak at 60° C. (see FIG. 2 ), indicating that there is no non-specific amplification.

Embodiment 2 Zika virus detection standard curve making

In this example, the sensitivity of the primers and probes of the one-step fluorescent rt-PCR detection method of the present invention is evaluated by using the Zika virus stock solution and the synthetic plasmid standard containing the nucleic acid fragment of the detection target gene.

1. Acquisition of plasmid standards

The plasmid standard is obtained by connecting the synthetic Zika virus gene target fragment DNA (see SEQ ID No.4) T/A cloning method into the vector pTG19-T, pick 8 clones, and shake the bacteria to extract the plasmid for sequencing verification , select the plasmid with the correct sequencing result as the stock solution of the plasmid standard. The plasmid concentration was detected by ultraviolet spectrophotometry, and the copy number of the standard was determined according to the following calculation formula: (ng/μL)×10 ^-9 ×6.02×10 ²³ /(bp×660)=copies/μL (where: 6.02×10 ²³ is the molar constant, and 660 is the base (AGCT) average molecular weight). Then the standard was diluted, specifically, the stock solution of the plasmid standard was diluted in a 10-fold gradient to 2.0×10 ¹ -2.0×10 ⁷ copies/mL, and a total of 7 concentration gradients were detected, with 3 replicates each.

2. Using plasmid standards to make a standard curve

Real Time One Step RT-PCR (Takara, RR064A) was used to monitor the fluorescence signal of the probe (SEQID NO.3).

The principle is as follows: the probe (SEQ ID NO.3) uses an oligonucleotide modified with a fluorescent substance (FAM) at the 5' end and a quencher substance (MGB) at the 3' end for fluorescence detection. When the probe is intact, the fluorescent substance at the 5' end is restricted by the quenching substance at the 3' end and cannot emit fluorescence. When the probe is decomposed, the fluorescent substance at the 5' end will dissociate and emit fluorescence. When the fluorescent probe is added to the PCR reaction solution, the fluorescent probe will hybridize with the template during the annealing process of the PCR reaction. Further, during the extension process of the PCR reaction, the 5→3' exonuclease activity of Taq DNA polymerase can decompose the fluorescent probe hybridized with the template, and the free fluorescent substance emits fluorescence. By detecting the fluorescence intensity in the reaction system, the purpose of detecting the amplification amount of the PCR product can be achieved.

Prepare the RT-PCR reaction solution according to the components in Table 4 (please prepare the reaction solution on ice).

Table 4 RT-PCR reaction system

Remark:

*1: The final concentration of the primer is preferably 0.1 to 1 μM. (Figure 3 and Figure 4 are the results of the final primer concentration of 0.2 μM)

*2: The final concentration of the probe is preferably 0.1 to 0.5 μM. (Figure 3 and Figure 4 are the results of the final probe concentration of 0.3 μM)

*3 Used to correct the fluorescence signal error between wells.

*4 In actual operation, the total system can be adjusted according to the reaction system recommended by the instrument.

The reaction conditions are as follows:

Stage 1: Reps: 1

42℃, 5min

95℃, 10sec

Stage 2: Reps: 40

95℃, 5sec

60°C, 34sec

The results are shown in Figure 3 and Figure 4. The plasmid standard was serially diluted 10-fold, with a total of 7 concentration gradients. Figure 3 is the amplification curve of the plasmid standard at each concentration. Its standard curve (see Figure 4) is obtained by fitting the Ct value and the plasmid copy concentration, the linear relationship of the standard curve is y=-3.3214x+34, R ² =0.99827, indicating that the present invention is 2.0×10 ⁷ ~ 2.0× There is a good linear relationship between 10 ¹ copies/mL, (where y is the ct value, which is the cycle threshold, x is the logarithm of the copy number of the plasmid standard product with base 10, and R is the correlation coefficient). The detection limit is about 20copies/mL.

Interpretation of the results: the Ct value of the sample amplification curve <34 is positive for Zika virus; the Ct value of the sample >34 is negative for Zika virus, and the copy number of Zika virus can be calculated for positive samples according to the standard curve.

The Ct value is determined as: the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value.

The fluorescence threshold is defined as: the fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal, and the default setting of the fluorescence threshold is 10 times the standard deviation of the fluorescence signal of 3 to 15 cycles.

The establishment and concrete operation of embodiment 3 kit

1. Kit composition

Prepare the test kit for detecting Zika virus according to the following composition: the upstream primer of embodiment 1, downstream primer, fluorescent probe, positive standard (i.e. the plasmid standard that embodiment 2 makes), negative quality control product RT- PCR reaction solution, reverse transcriptase, PCR enzyme. The RT-PCR reaction liquid consists of four nucleotides, dATP, dUTP, dGTP, and dCTP, and a buffer containing magnesium ions. The negative quality control substance is sterile DEPC water.

2. Detection steps

(1) PCR reaction

Using the sample to be tested (viral RNA) as a template, prepare the RT-PCR reaction system according to Table 5

Table 5 RT-PCR reaction system

Remark:

*1: The final concentration of the primer is preferably 0.1 to 1 μM, more preferably 0.2 μM.

*2: The final concentration of the probe is preferably 0.1 to 0.5 μM, more preferably 0.3 μM.

*3 In actual operation, the total system can be adjusted according to the reaction system recommended by the instrument.

The RT-PCR reaction system may also include a fluorescence signal correction reagent, such as ROX Reference Dye or DyeII (50×), which can be used to correct the fluorescence signal error between wells.

The reaction conditions of PCR were: reverse transcription at 42°C for 5min, 95°C, pre-denaturation for 10s; denaturation at 95°C for 5s, fluorescence detection at 60°C for 34s, 40 cycles.

(2) Interpretation of results: The Ct value of the sample amplification curve <34 is positive for Zika virus; the Ct value of the sample >34 is negative for Zika virus, and the copy number of Zika virus can be calculated for positive samples according to the standard curve.

Example 4 Detection of Zika virus Asian type and specificity test

The specificity of the optimal primers of the present invention was detected by using Zika virus Asian type (KU955589.1) cell culture fluid.

1. Extraction of viral RNA

According to the reagent manufacturer's instructions, the ZIKV virus nucleic acid in the Zika virus (KU955589.1) cell culture medium was extracted by TRIZOL (Takara, 9109) nucleic acid extraction method. The positive quality control powder was briefly centrifuged and dissolved in 0.5 mL sterile DEPC water. The extraction method of positive quality control virus RNA is the same as above. The positive quality control products are dengue type 1 strain (KM204119), dengue type 2 strain (KF479233), dengue type 3 strain (AY744683), dengue type 4 strain (KJ596658), Japanese B A mixture of type meningitis virus strain (KF667322) and influenza A virus strain (A/FM/1/4/) inactivated strain (the above viruses were provided by the Department of Pathogenic Microbiology and Immunology, Jinan University).

For specific steps, see "1. Extracting viral RNA" in Example 1.

2. Using the detection method constructed in Example 3, that is, the one-step probe method, Real Time One Step RT-PCR (Takara, RR064A) was used to monitor the fluorescence signal of the probe (SEQ ID NO.3).

Prepare the RT-PCR reaction solution according to Table 6 (please prepare the reaction solution on ice).

Table 6 RT-PCR reaction system

Remark:

*1 The final concentration of the primer is preferably 0.1 to 1 μM. (Figure 5 is the result of the final primer concentration of 0.2 μM)

*2 The final concentration of the probe is preferably 0.1 to 0.5 μM. (Figure 5 is the result of the final probe concentration of 0.3 μM)

*3 Used to correct the fluorescence signal error between wells.

*4 In actual operation, the total system can be adjusted according to the reaction system recommended by the instrument.

The reaction conditions are as follows:

Stage1: Reps: 1

42℃, 5min

95℃, 10sec

Stage 2: Reps: 40

95℃, 5sec

60°C, 34sec

The results of the amplification curve are shown in Figure 5, A is the detection sample of the Asian strain of Zika virus, and B is the positive quality control product. The Ct values of A are all less than 34; while the Ct values of the positive quality controls are all greater than 34. It can be seen that the present invention is applicable to the detection of Asian strains of Zika virus, and does not cross-react with other viruses of the genus Yellow fever, does not cross-react with other types of viruses and cell substrates, and shows good specificity.

Example 5 Detection of Zika virus African type and specificity test

The specificity of the optimal primers of the present invention was detected by using Zika virus African type (DQ859059) (provided by the Department of Pathogenic Microbiology and Immunology of Jinan University) cell culture fluid.

1. Extraction of viral RNA

According to the reagent manufacturer's instructions, TRIZOL (Takara, 9109) nucleic acid extraction method was used to extract the ZIKV virus nucleic acid in the Zika virus (DQ859059) cell culture medium. Wherein the powder of the positive quality control product (same as in Example 4) was briefly centrifuged and dissolved in 0.5 mL of sterile DEPC water.

The specific steps are the same as "1. Extracting viral RNA" in Example 1.

2 Real Time One Step RT-PCR (Takara, RR064A) was used to monitor the fluorescence signal of the probe (SEQID NO.3) by one-step probe method.

Prepare the RT-PCR reaction solution according to the components in Table 5 (please prepare the reaction solution on ice).

Reaction condition is with embodiment 4.

The results of the amplification curve are shown in Figure 6, A is the test sample of Zika virus African type strain, and B is the positive quality control product. The Ct values of A are all less than 34; while the Ct values of the positive quality controls are all greater than 34. It can be seen that the present invention is applicable to the detection of African strains of Zika virus, and does not cross-react with other viruses of the genus Yellow fever, does not cross-react with other types of viruses and cell substrates, and shows good specificity.

Embodiment 6 applicability test

Serum samples isolated from two Zika virus patients were selected (serum from confirmed Zika patients were provided by the Guangdong Provincial Center for Disease Control), and Zika virus detection was performed according to the method in Example 3 to verify the applicability of the kit.

1. Extraction of viral RNA

According to the reagent manufacturer's instructions, TRIZOL (Takara, 9109) nucleic acid extraction method was used to extract the ZIKV virus nucleic acid in the serum of Zika patients. Wherein the powder of the positive quality control product (same as in Example 4) was briefly centrifuged and dissolved in 0.5 mL of sterile DEPC water. The specific steps are the same as "1. Extracting viral RNA" in Example 1.

2. Real Time One Step RT-PCR (Takara, RR064A) was used to monitor the fluorescent signal of the probe (SEQ ID NO.3) by one-step probe method.

Prepare the RT-PCR reaction solution according to the components in Table 5 (please prepare the reaction solution on ice).

Reaction condition is with embodiment 4.

The results of the amplification curves are shown in Figure 7. A is a test sample of a patient virus strain, B is a test sample of another patient virus strain, and C is a positive quality control product. The Ct values of A and B are both less than 34, which are judged as positive, while the Ct values of the positive quality control products are both greater than 34. It can be seen that the present invention can detect clinical blood samples and has good applicability.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

sequence listing

<110> Jinan University

<120> One-step fluorescent rt-PCR detection method and kit for Zika virus

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<170> SIPOSequenceListing 1.0

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ctgataacat caccttggca atcctggctg ctctgacacc actggcccgg ggcacactgc 180

ttgtggcgtg gagagcaggc cttgctactt gcggggggtt tatgctcctc tctctgaagg 240

gaaaaggcag tgtgaagaag aacttaccat ttgtcatggc cctgggacta accgctgtga 300

ggctggtcga ccccatcaac gtggtgggac tgctgttgct cacaaggagt gggaagcgga 360

gctggccc 368

Claims (10)

1. A genetic marker for detecting Zika virus, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.4. 2. the application of the genetic marker for detecting Zika virus described in claim 1 in the preparation detection kit of Zika virus. 3. A pair of specific primers for detecting Zika virus, characterized in that: The upstream primer sequence is: 5'-GGTTCTCATCAATGGTTTTGCT-3'; The downstream primer sequence is: 5'-TGGAACAAACCATCGCTCGTA-3'. 4. A fluorescent probe for use in conjunction with the specific primer as claimed in claim 3, characterized in that: Its sequence is 5'-TGGCCTGGTTGGCA-3', a fluorescent reporter group is marked at the 5' end of the sequence, and a fluorescent quencher group is marked at its 3' end. 5. the application of the specific primer pair for detecting Zika virus described in claim 3 and/or the fluorescent probe described in claim 4 in the preparation detection kit of Zika virus. 6. A detection kit for Zika virus, characterized in that: Containing the specific primer pair for detecting Zika virus described in claim 3. 7. detection Zika virus kit according to claim 6, is characterized in that: Also comprise the fluorescent probe described in claim 4. 8. A Zika virus one-step fluorescence rt-PCR detection method, is characterized in that: Real-time fluorescence quantitative PCR is carried out by using the specific primer pair shown in SEQ ID NO.1, SEQ ID NO.2 and the fluorescent probe shown in SEQ ID NO.3 to detect Zika virus in the sample to be tested. 9. Zika virus one-step fluorescent rt-PCR detection method according to claim 8, is characterized in that: In the fluorescent rt-PCR reaction system, the final concentration of the primer is 0.1-1 μM; the final concentration of the fluorescent probe is 0.1-0.5 μM. 10. Zika virus one-step fluorescent rt-PCR detection method according to claim 8, is characterized in that: The real-time fluorescent quantitative PCR reaction conditions are: reverse transcription at 42°C for 5 min, 95°C, pre-denaturation for 10 s; denaturation at 95°C for 5 s, fluorescence detection at 60°C for 34 s, and 40 cycles.