Congo fever virus Taqman probe fluorescent quantitative PCR detection kit and application thereof

Abstract

The invention discloses a Congo fever virus Taqman probe fluorescent quantitative PCR (polymerase chain reaction) detection kit and application thereof, specific primers and corresponding fluorescent probes of the Congo fever virus are designed, and then a conserved gene sequence of the virus is selected by utilizing a known virus genome sequence; the method comprises the following steps: constructing a standard recombinant plasmid containing a conserved gene sequence by using a gene cloning technology, then identifying the recombinant plasmid to prepare a plasmid standard substance so as to obtain a standard curve, evaluating the sensitivity, specificity and repeatability of a PCR (Polymerase Chain Reaction) method, and finally forming the detection kit by using a fluorescent quantitative PCR reaction system. The kit is simple and efficient in detection operation, can realize rapid detection of the Congo fever virus, has the characteristics of high sensitivity, strong specificity and good repeatability, and can be widely applied to clinical nucleic acid sample detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and relates to a Taqman probe fluorescence quantitative PCR detection kit for Crimean Congo hemorrhagic fever virus and its application. Background Art

[0002] Crimean Congo hemorrhagic fever virus (CCHFV), also known as Crimean-Congo hemorrhagic fever virus, is a highly contagious tick-borne virus classified as a biosafety level 4 virus. It belongs to the order Bunyavirales, family Nairoviridae, genus Orthonairovirus, and mainly forms a transmission chain of tick→animal→human and tick→human→human. In this process, infected animals only have viremia, but infected humans will show severe bleeding and clinical symptoms accompanied by multi-organ damage. The clinical manifestations mainly include headache, fever, vomiting, bleeding, shock and other symptoms, and can lead to death in severe cases, with a mortality rate of about 10%-40%. Due to its potential epidemicity, high mortality rate and difficulties in treatment and prevention, CCHFV will pose a threat to China's public health security.

[0004] At present, the laboratory diagnosis of CCHFV mainly includes serological diagnosis, molecular biology methods and virus culture. Serological detection techniques mainly include indirect immunofluorescence assay, enzyme-linked immunosorbent assay and immunochromatography. However, the indirect immunofluorescence assay has certain requirements for laboratory conditions, and the operation process is relatively cumbersome, requiring professional personnel; the enzyme-linked immunosorbent assay requires specific equipment, and the operation process needs to be strictly controlled to avoid false positive or false negative results; the immunochromatography has low sensitivity and specificity. Molecular biology detection techniques mainly include quantitative real-time PCR (qPCR) and nucleic acid sequencing, etc., but they require expensive and sophisticated equipment, and the operation process is complex, with high costs and long time consumption, which is not suitable for clinical rapid diagnosis. And virus culture has high requirements for laboratory conditions and long time consumption.

[0005] TaqMan fluorescent quantitative PCR technology is a highly specific and sensitive molecular biology technology, which is widely used in gene expression analysis, pathogen detection, mutation analysis, drug development and other fields. Its core lies in the design of specific probes. The 5' end of the probe is labeled with a reporter fluorescent group, and the 3' end is labeled with a fluorescent quencher group. When the probe is intact, the fluorescent energy emitted by the reporter fluorescent group is absorbed by the quencher group, and the instrument cannot detect the fluorescent signal. During the polymerase chain reaction (PCR) reaction, Taq DNA polymerase synthesizes a new DNA chain from 5' to 3' on the template chain. Its 5'→3' exonuclease activity will cut the reporter fluorescent group at the 5' end of the probe, making it free from the reaction system and free from the shielding of the 3' end fluorescent quencher group, so as to receive the fluorescent signal. After each PCR cycle, the fluorescent signal will grow synchronously, realizing the synchronization of fluorescent signal accumulation and PCR product. Its intensity represents the copy number of template DNA, so this technology can accurately quantify the template. TaqMan fluorescent quantitative PCR technology has a closed reaction system, which can ensure the high specificity of the reaction; its high sensitivity and precise quantitative ability can detect extremely low concentrations of target nucleic acid sequences; it can monitor the progress of the PCR reaction in real time, so as to obtain the results in time; and it can detect multiple target genes or sequences in the same reaction. Therefore, Taqman fluorescent quantitative PCR technology is simple to operate, efficient, and has the characteristics of high sensitivity, strong specificity, and good repeatability, and can be widely used in clinical sample detection. However, there is no report on the application of Taqman fluorescent quantitative PCR technology for the detection and diagnosis of CCHFV. Summary of the invention

[0006] The purpose of the present invention is to overcome the above technical problems existing in the existing detection methods of CCHFV, and to provide a Congo fever virus Taqman probe fluorescent quantitative PCR detection kit with high sensitivity, strong specificity and good repeatability through methods such as primer design, plasmid construction and identification, and standard curve acquisition.

[0007] Another object of the present invention is to provide the use of the above-mentioned Congo fever virus Taqman probe fluorescent quantitative PCR detection kit in clinical nucleic acid sample detection.

[0008] To achieve its purpose, the present invention adopts the following technical solution:

[0009] The present invention provides a Congo fever virus Taqman probe fluorescent quantitative PCR detection kit, comprising a specific qPCR detection primer pair for Congo fever virus and a TaqMan probe, wherein the specific qPCR detection primer pair comprises a forward primer and a reverse primer; wherein:

[0010] The forward and reverse primer sequences for detecting Crimean-Congo hemorrhagic fever virus are shown in SEQ ID NO.1-2; the Taqman probe sequence is shown in SEQ ID NO.3.

[0011] As a further preference of the technical solution of the present invention, the kit further comprises: Premix Ex Taq (Probe qPCR) (2X), Crimean-Congo hemorrhagic fever virus DNA template and sterilized water.

[0012] Furthermore, the kit contains 0.4 μL of each of the forward and reverse primers for Crimean-Congo hemorrhagic fever virus, 0.8 μL of Taqman probe, 10 μL of Premix Ex Taq (Probe qPCR) (2X), 2 μL of Crimean-Congo hemorrhagic fever virus DNA template, and 6.4 μL of sterilized water.

[0013] Furthermore, the optimal concentrations of the forward and reverse primers in the kit are 0.1 μM, and the concentration of Taqman probe is 0.1 μM.

[0014] Furthermore, the fluorescence quantitative PCR reaction procedure is as follows:

[0015] Pre-denaturation at 95 °C for 30 s, 1 cycle; denaturation at 95 °C for 5 s, annealing and extension at 62 °C for 30 s, for 45 cycles; melting curve analysis: 50 °C, 30 s, 1 cycle; reaction ends.

[0016] Furthermore, the lowest copy number of Crimean-Congo hemorrhagic fever virus that the kit can detect is 10 copies / μL.

[0017] The above-mentioned fluorescence quantitative PCR detection kit for Crimean-Congo hemorrhagic fever virus Taqman probe can be used for clinical nucleic acid sample detection.

[0018] Compared with the prior art, the fluorescence quantitative PCR detection kit for Crimean-Congo hemorrhagic fever virus Taqman probe provided by the present invention has the following beneficial effects:

[0019] 1. It provides a simple, efficient and low-cost method for detecting Crimean-Congo hemorrhagic fever virus.

[0020] 2. The present invention is characterized by high sensitivity, strong specificity and good repeatability.

[0021] 3. The kit of the present invention can be used for clinical nucleic acid sample detection, so as to quickly diagnose and monitor Crimean-Congo hemorrhagic fever virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the identification result of CCHFV-N gene recombinant enzyme digestion;

[0023] Figure 2Results of CCHFV-N protein expression identification;

[0024] Figure 3 CCHFV-N standard curve;

[0025] Figure 4 Results of CCHFV-N sensitivity experiment;

[0026] Figure 5 Results of CCHFV-N qPCR specificity experiment. Specific implementation mode

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings through specific embodiments.

[0028] Example 1: Obtaining specific primers and probes

[0029] Query the publicly available complete genes of CCHFV on NCBI GeneBank. After downloading, perform sequence analysis on the complete genes using MEGA7.0 software. Through the analysis results and literature review, relatively conserved target genes (N, F, G) are screened out. Then, use MEGA7.0 to perform sequence alignment on the target genes to find the core conserved regions. Select the N gene of Congo hemorrhagic fever virus (CCHFV-N), perform sequence alignment on the target gene using MEGA7.0 to find the core conserved region. Use Oligo7 software to design primer sequences and probe sequences for the conserved gene N gene of CCHFV, and verify their specificity through Blast. The primers and probes are synthesized by BGI. The specific primer sequences and the nucleotide sequences of the corresponding Taqman fluorescent probes are shown in Table 1 respectively.

[0030] Table 1 CCHFV-N primer and Taqman probe sequences

[0031]

[0032] Example 2: Identification of CCHFV-N gene recombinant plasmid

[0033] According to the primers and target gene sequences selected in Example 1, they are synthesized by BGI to construct a recombinant plasmid.

[0034] Transformation, amplification and extraction of recombinant plasmid:

[0035] 1. Transformation of recombinant plasmid

[0036] Take 2 μL of the recombinant plasmid and add it to an EP tube containing 50 μL of DH5α competent cells. Gently pipette up and down to mix well. Place it on ice for 30 min, heat shock at 42 °C for 45 s, and then place it on ice for 2 - 3 min. Add 900 μL of antibiotic-free LB liquid medium, and shake the bacteria at 37 °C and 220 rpm on a constant temperature shaker for 1 h. Take out the shaken EP tube, centrifuge at 5000 rpm for 5 min, discard the supernatant, resuspend the bacterial cells with the remaining medium, and use a sterile spreader to evenly spread the bacterial cells on a solid LB medium plate with the corresponding antibiotic resistance. Incubate it upside down in an incubator at 37 °C for 12 h.

[0037] 2. Amplification of the recombinant plasmid

[0038] Pick a single colony from the LB plate and inoculate it into 5 mL of liquid LB medium. Incubate it at 37 °C and 220 rpm on a constant temperature shaker for 12 h.

[0039] 3. Extraction of the recombinant plasmid

[0040] Transfer the bacterial liquid cultured for 12 h in step 2 to a clean 2 mL centrifuge tube, centrifuge at 11000 rpm for 1 min at room temperature, and discard the waste liquid. Use the Omega plasmid miniprep Kit I. According to the operation manual, add 250 μL of Solution I, vortex and shake thoroughly until there is no obvious bacterial cell precipitate in the tube. Add 250 μL of Solution II, gently mix, and let it stand for 2 - 3 min. Add 350 μL of Solution III, gently mix (a milky white flocculent substance appears), and centrifuge at 12000 rpm for 10 min. Gently transfer the supernatant to the adsorption column provided in the kit, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Add approximately 500 μL of HBC Buffer to the same adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Add approximately 700 μL of DNA Wash Buffer to the same adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Repeat adding 700 μL of DNA Wash Buffer, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Centrifuge the adsorption column at 12000 rpm for 2 min without sample. Open the lid and let it stand to volatilize ethanol. Transfer the adsorption column to a new 1.5 mL centrifuge tube. Add 35 μL of sterile ddH2O (deionized water) to the center of the column membrane, cover the lid and let it stand for 1 min, centrifuge at 12000 rpm for 1 min, and store it at 4 °C. Measure the concentration of the extracted plasmid using a ultra-micro spectrophotometer.

[0041] 4. Identification of the recombinant plasmid

[0042] (1) Enzyme digestion identification of the recombinant plasmid:

[0043] The extracted CCHFV-N gene plasmid was digested with restriction enzymes EcoRⅠ and XhoⅠ. The digestion products were subjected to agarose gel electrophoresis. The bottom band was the CCHFV-N gene fragment, and the fragment size was consistent with the expectation. It was digested into two fragments of 330 bp and 1157 bp; the results were as Figure 1 shown.

[0044] (2) Identification of recombinant plasmid protein expression

[0045] The synthesized CCHFV-N gene plasmid was transfected into 293T cells. The culture conditions were DMEM + 10% FBS + 1% P / S, and the culture environment was 5% carbon dioxide (CO2) at 37 °C. After 18 h, the cell protein samples were collected. The protein immunoblotting experiment was used to verify the expression of CCHFV-N protein. The expression of CCHFV-N protein was correct. The results were as Figure 2 shown.

[0046] Example 3: Preparation of CCHFV-N gene recombinant plasmid standard

[0047] According to the size and concentration of the recombinant plasmid, the copy number of the plasmid in the plasmid stock solution was calculated according to the following formula:

[0048]

[0049] Y represents the copy number, and X represents the measured plasmid concentration in ng / μL.

[0050] The size of the CCHFV-N gene plasmid was 6843 bp, and the concentration was 504.7 ng / μL. The calculated copy number was 6.727×10 10 copies / μL.

[0051] The CCHFV target gene recombinant plasmid stock solution was serially diluted with TE Buffer to concentrations of 1×10 0 、1×10 1 、1×10 2 、1×10 3 、1×10 4 、1×10 5 、1×10 6 、1×10 7 、1×10 8 、1×10 9 、1×10 10 copies / μL of standards. After aliquoting, they were stored at -80 °C in the refrigerator for later use.

[0052] Example 4: Construction and optimization of TaqMan qPCR detection method

[0053] Using the CCHFV target gene plasmid standard with a concentration of 1×10 7 copies / μL as a template, Premix Ex TaqTM (Probe qPCR) was used to construct a single TaqMan qPCR detection method. Three replicates of the CCHFV target gene plasmid standard were set up, and three negative controls were set. The reaction system was prepared as follows: Premix Ex Taq (Probe qPCR) (2X) 10 μL; forward primer (10 μM) 0.4 μL; reverse primer (10 μM) 0.40 μL; Probe probe (10 μM) 0.8 μL; DNA template 2.0 μL; ddH2O 6.4 μL; the total system was 20 μL. After mixing the system, the solution was added to a 96-well plate, sealed with a film, and reacted on a LightCycler 480 System. The qPCR reaction conditions were: pre-denaturation: 95°C, 30 s, 1 cycle; PCR: 95°C, 5 s, 60°C, 30 s for a total of 45 cycles, and fluorescence was collected; melting curve analysis: 50°C, 30 s, 1 cycle.

[0054] Based on the established qPCR method, using the CCHFV target gene plasmid standard with a concentration of 1×10 7 copies / μL as a template, with the probe amount fixed, the usage amounts of the forward and reverse primers with a concentration of 10 μM were 0.5, 1.0, 1.5, 2.0, and 2.5 μL respectively; with the primer amount fixed, the usage amounts of the probe with a concentration of 10 μM were 0.5, 1.0, 1.5, 2.0, and 2.5 μL; and negative controls and blank controls were set. The blank group was replaced with sterile ddH2O, and parallel detection was performed 3 times. Using the Ct value and fluorescence intensity as the judgment criteria, the optimal primer and probe concentrations were determined.

[0055] Based on the established qPCR method, using the CCHFV target gene plasmid standard with a concentration of 1×10 7 copies / μL as a template, using the optimal combination of primers and probes, the annealing temperature of the TaqMan qPCR detection method was optimized. The temperature range was set from 58 - 66°C, namely 58, 60, 62, 64, and 66°C. Negative controls and blank controls were set for each temperature gradient. By comparing the Ct value, fluorescence intensity, and whether non-specific amplification occurred, the optimal annealing temperature was determined.

[0056] The experiment obtained that the optimal annealing temperature of the optimized CCHFV-N reaction system was 62°C. The optimal forward / reverse primer concentration was 0.1 μM, and the probe concentration was 0.1 μM (Ct value = 17.87 ± 0.1). The results are shown in Table 2.

[0057] Table 2 Ct values of single qPCR of CCHFV-N at different probe and primer concentrations

[0058]

[0059] Example 5: Establishment of TaqMan qPCR Standard Curve

[0060] The aliquoted plasmid standard of CCHFV-N gene with different concentrations was diluted to 1.0×10 10 copies / μL with TE Buffer, and then serially diluted 10-fold to prepare standards with concentrations of 10 0 -10 10 copies / μL. Using the above standards as templates, under the optimal qPCR reaction conditions determined previously, Premix Ex TaqTM (Probe qPCR) was used for singleplex qPCR detection and the standard curve was plotted. The CCHFV-N standard curve obtained from the figure was: Y = -3.364X + 39.748; R 2 = 0.9986, indicating a good correlation between the logarithm of the template amount at different concentration gradients and the Ct value; the amplification efficiency E = 98.27%, indicating that the qPCR method had a good amplification efficiency. The CCHFV-N standard curve is as Figure 3 shown.

[0061] Example 6: Sensitivity Test of Singleplex qPCR

[0062] The aliquoted plasmid standard of CCHFV target gene with a concentration of 10 10 -10 0 copies / μL was used as a template, and qPCR detection was performed under the reaction conditions determined previously. With Ct value ≤ 35 as the threshold, the sensitivity of the method was detected. The lowest copy number of the CCHFV-N gene that could be detected was 10 copies / μL. It was concluded that its sensitivity was relatively high, as Figure 4 shown.

[0063] Example 7: Specificity Test of Singleplex qPCR

[0064] Based on the preliminary evaluation of primer and probe specificity by NCBI Blast, this method was used to detect gene plasmids of Nipah virus (NiV-N), Rift Valley fever virus (RVFV-N), and Hendra virus (HeV-N). Positive nucleic acids of N genes of other viruses stored in this laboratory, including avian influenza virus (AIV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), Sendai virus (SeV), etc., were selected as samples to be tested. At the same time, a negative (sterile water) control was set up, and qPCR detection was performed using the established detection method to evaluate the specificity of this method. The results showed that the results of positive nucleic acids of other viruses were all negative, and only the CCHFV-N gene plasmid group showed an amplification curve, indicating that this method has high specificity, as Figure 5 shown.

[0065] Example 8: Singleplex qPCR reproducibility test

[0066] Taking standard products of CCHFV target gene plasmids with different concentrations as templates, qPCR reactions were carried out with parallel wells set for 9 different concentration gradients with a concentration of 10 10 -10 2 copies / μL. The Ct value of each group was recorded as the within-run repeat test; after an interval of three days, the same batch of standards stored in a -80°C refrigerator was used again for qPCR reaction, and the Ct value of each group was recorded as the between-run repeat experiment. The mean (M), standard deviation (SD), and coefficient of variation (CV) of the nucleic acid amplification Ct values of standard products with different concentrations were calculated respectively, and the reproducibility of this method was evaluated according to the results of the two repeat tests. The results are shown in Table 3.

[0067] Table 3 Ct values of CCHFV-N qPCR parallel experiments

[0068]

[0069] Note: Copies, copy number; Average, average value; SD = standard deviation, standard deviation; CV = Coefficient of Variation, coefficient of variation.

[0070] The results in Table 3 showed that the coefficient of variation < 2%, indicating that the method has good reproducibility and stable results.

Claims

1. A TaqMan probe fluorescence quantitative PCR detection kit for Congo fever virus, characterized in that, The kit includes a specific qPCR detection primer pair and a TaqMan probe for Crimean-Congo hemorrhagic fever virus. The specific qPCR detection primer pair includes a forward primer and a reverse primer; wherein: The sequences of the forward primer and the reverse primer for detecting Crimean-Congo hemorrhagic fever virus are shown in SEQ ID NO.1-2; the sequence of the Taqman probe is shown in SEQ ID NO.

3.

2. The TaqMan probe fluorescence quantitative PCR detection kit for Crimean-Congo hemorrhagic fever virus according to claim 1, wherein The kit further includes: Premix Ex Taq (Probe qPCR) (2X), Crimean-Congo hemorrhagic fever virus DNA template and sterilized water.

3. The TaqMan probe fluorescence quantitative PCR detection kit for Congo fever virus according to claim 2, wherein, The kit contains 0.4 μL of each of the forward and reverse primers for Crimean-Congo hemorrhagic fever virus, 0.8 μL of TaqMan probe, 10 μL of Premix Ex Taq (Probe qPCR) (2X), 2 μL of Crimean-Congo hemorrhagic fever virus DNA template, and 6.4 μL of sterilized water.

4. The TaqMan probe fluorescence quantitative PCR detection kit for Crimean-Congo hemorrhagic fever virus according to claim 3, characterized in that, The concentrations of the forward and reverse primers in the kit are 0.1 μM, and the concentration of the TaqMan probe is 0.1 μM.

5. The TaqMan probe fluorescence quantitative PCR detection kit for Congo fever virus according to claim 3, wherein, The fluorescence quantitative PCR reaction procedure is as follows: Pre-denaturation at 95°C for 30 s, 1 cycle; denaturation at 95°C for 5 s, annealing and extension at 62°C for 30 s, for 45 cycles; melting curve analysis: 50°C, 30 s, 1 cycle; reaction ends.

6. The Taqman probe fluorescence quantitative PCR detection kit for Congo fever virus according to claim 4, characterized in that, The lowest copy number of Crimean-Congo hemorrhagic fever virus that the kit can detect is 10 copies / μL.

7. Use of a TaqMan probe fluorescence quantitative PCR detection kit for Crimean-Congo hemorrhagic fever virus according to any one of claims 1-6 in the detection of clinical nucleic acid samples.

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