Taqman probe fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for Hendea virus and application of Taqman probe fluorescent quantitative PCR detection kit

Abstract

The invention discloses a Hendea virus Taqman probe fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit and application thereof, specific primers and corresponding fluorescent probes of the Hendea 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 Hendea 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 invention relates to the field of biotechnology, and relates to a Hendra virus Taqman probe fluorescent quantitative PCR detection kit and an application thereof. Background Art

[0002] Hendra virus (HeV), formerly known as equine measles virus, is a highly lethal zoonotic virus and is classified as a biosafety level 4 virus. It is classified as a Paramyxoviridae and Henipavirus genus. Compared with other Paramyxoviridae viruses, HeV has a wide host range, no hemagglutination and no neuraminidase activity. HeV first broke out in a suburb of Brisbane, Queensland, Australia in 1994, killing 14 racehorses and 2 people. Fruit bats of the Pteropus family are considered to be the natural hosts of HeV, and they are infected or carry the virus but do not show symptoms. In Australia, more than a quarter of fruit bats can be tested positive for Hendra virus-specific antibodies and the virus can be isolated. Studies have shown that HeV may only naturally infect fruit bats, humans and horses, and can also infect susceptible animals such as dogs, cats, and guinea pigs through experiments. The transmission route of HeV is not yet completely clear, but the excreta of infected animals contains a high level of virus, which should be the main route of detoxification of infected animals. The virus can also be transmitted through direct contact with saliva or nasal mucus. Studies have shown that direct transmission between humans is not easy, but feeding infected horses, direct contact with infected horses, or contact with fluids and tissues contaminated by the virus may cause human infection. Human infection with HeV can lead to fatal respiratory or encephalitis and widespread vascular inflammation. According to surveys, its mortality rate can reach 40% to 75%.

[0003] 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 that ensures 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 to obtain results in a timely manner; and it can detect multiple target genes or sequences in the same reaction. TaqMan fluorescent quantitative PCR technology plays an important role in life science research and clinical diagnosis with its high efficiency, specificity and sensitivity.

[0004] HeV infection has a short course, high mortality rate, and wide species tropism. Once spread, it will bring a major blow to local public health, animal husbandry safety and economic development. The current prevention and control measures in my country are to strengthen the quarantine of animals and related products at ports to prevent the import of epidemics, and to kill infected animals immediately once confirmed. HeV is one of the mandatory inspection items for horses entering and leaving the country by my country's customs, and it occupies an important position in detection and diagnosis, so it is crucial to develop a broad-based multi-faceted diagnostic strategy. Currently, the commonly used laboratory diagnostic techniques for HeV include: pathogen detection, immunological detection, and molecular biological detection. Initially, serum neutralization experiments were used for preliminary monitoring of HeV, but most infected animals died before an effective immune response was produced, so this method is not suitable for the initial detection of HeV infection. Pathogenic diagnosis is the gold standard for clinical diagnosis of diseases, and isolation and cultivation of HeV from infected animals can assist diagnosis to the greatest extent. However, due to the limiting factors such as HeV's high requirements for laboratory infrastructure, operator proficiency, and slow and difficult pathogen isolation, it is currently difficult for many regions to use virus isolation and cultivation methods to meet clinical detection needs. ELISA is low-cost, highly specific, and highly sensitive, and is widely used in clinical testing, but it has low specificity and leads to frequent false positives and nonspecific reactions. Currently, there are no reports on the use of Taqman fluorescent quantitative PCR technology for the detection and diagnosis of HeV. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned technical problems existing in the existing HeV detection methods, and to provide a Hendra 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.

[0006] Another object of the present invention is to provide the use of the above-mentioned Hendra virus Taqman probe fluorescent quantitative PCR detection kit in clinical nucleic acid sample detection.

[0007] To achieve its purpose, the present invention adopts the following technical solution:

[0008] The present invention provides a Hendra virus Taqman probe fluorescent quantitative PCR detection kit, comprising a specific qPCR detection primer pair for Hendra virus and a TaqMan probe, wherein the specific qPCR detection primer pair comprises a forward primer and a reverse primer; wherein:

[0009] The forward primer and reverse primer sequences for detecting Hendra virus are shown in SEQ ID NO.1-2; the Taqman probe sequence is shown in SEQ ID NO.3.

[0010] As a further preferred embodiment of the technical solution of the present invention, the kit also includes: Premix Ex Taq (ProbeqPCR) (2X), Hendra virus DNA template and sterile water.

[0011] Furthermore, the kit contains 0.4 μL each of Hendra virus forward and reverse primers, 0.8 μL of Taqman probe, 10 μL of Premix Ex Taq (Probe qPCR) (2X), 2 μL of Hendra virus DNA template, and 6.4 μL of sterile water.

[0012] Furthermore, the optimal forward and reverse primer concentrations in the kit are 0.1 μM, and the Taqman probe concentration is 0.1 μM.

[0013] Furthermore, the fluorescence quantitative PCR reaction procedure is as follows:

[0014] Pre-denaturation at 95°C for 30 s, 1 cycle; denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, 45 cycles; melting curve analysis: 50°C for 30 s, 1 cycle; the reaction is complete.

[0015] Furthermore, the minimum copy number of Hendra virus that can be detected by the kit is 10 copies / μL.

[0016] The above-mentioned Hendra virus Taqman probe fluorescent quantitative PCR detection kit can be used for clinical nucleic acid sample detection.

[0017] Compared with the prior art, the Hendra virus Taqman probe fluorescent quantitative PCR detection kit provided by the present invention has the following beneficial effects:

[0018] 1. It provides a simple, efficient and low-cost method for Hendra virus detection.

[0019] 2. The present invention has the characteristics of high sensitivity, strong specificity and good repeatability.

[0020] 3. The kit of the present invention can be used for clinical nucleic acid sample detection, thereby quickly diagnosing and monitoring Hendra virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of HEV-N gene recombination enzyme digestion identification;

[0022] Figure 2 This is the result of HEV-N protein expression identification;

[0023] Figure 3 is the HEV-N standard curve;

[0024] Figure 4The results of the HEV-N sensitivity test;

[0025] Figure 5 The results are specific for HEV-N qPCR. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below through specific embodiments in conjunction with the accompanying drawings.

[0027] Example 1: Acquisition of specific primers and probes:

[0028] Search the NCBI GeneBank for the currently published full gene of HeV, download it, and perform sequence analysis on the full gene using MEGA7.0 software. By analyzing the results and consulting the literature, relatively conservative target genes (N, F, G) were screened out. Then use MEGA7.0 to perform sequence alignment on the target gene to find the core conservative region. Select the Hendra virus N gene (Hendravirus N gene, HeV-N) and use MEGA7.0 to perform sequence alignment on the target gene to find the core conservative region. Using Oligo7 software and referring to relevant literature, design the primer sequence and probe sequence of the HeV conserved gene N gene, and verify it by Blast to determine its specificity. The primers and probes were synthesized by BGI. The specific primer sequences and the corresponding Taqman fluorescent probe nucleotide sequences are shown in Table 1:

[0029] Table 1 HeV-N primers and Taqman probe sequences

[0030]

[0031] Example 2: Identification of HeV-N gene recombinant plasmid

[0032] The primers and target gene sequences selected in Example 1 were delivered to BGI for synthesis to construct a recombinant plasmid.

[0033] Transformation, amplification and extraction of recombinant plasmids:

[0034] 1. Transformation of recombinant plasmid

[0035] Take 2 μL of recombinant plasmid and add it to the EP tube containing 50 μL of DH5α competent cells, and gently blow to mix. Place on ice for 30 minutes, heat shock at 42℃ for 45 seconds, and place on ice for 2-3 minutes. Add 900 μL of LB liquid medium without resistance, and shake the bacteria on a constant temperature shaker at 37℃ and 220rpm for 1 hour. Take out the shaking EP tube, centrifuge at 5000rpm for 5 minutes, discard the supernatant, resuspend the bacteria with the remaining culture medium, and use a sterile coating rod to evenly spread the bacteria on the solid LB culture medium plate with the corresponding resistance. Incubate inverted at 37℃ for 14 hours.

[0036] 2. Amplification of recombinant plasmid

[0037] A single colony on the LB plate was picked and inoculated into 5 mL of liquid LB medium, and cultured on a constant temperature shaker at 37°C and 220 rpm for 14 h.

[0038] 3. Extraction of recombinant plasmid

[0039] Transfer the bacterial solution cultured for 12 hours in step 2 to a clean 2mL centrifuge tube, centrifuge at 11000rpm for 1min at room temperature, and discard the waste liquid. Use the Omega Plasmid Extraction Type I Kit according to the operating instructions, add 250μL SolutionⅠ, vortex and shake, and mix thoroughly until there is no obvious bacterial precipitation in the tube. Add 250μL SolutionⅡ, mix gently, and let stand for 2-3min. Add 350μL SolutionⅢ, mix gently (milky white flocculent material appears), and centrifuge at 12000rpm for 10min. Gently transfer the supernatant to the adsorption column provided with the kit, centrifuge at 12000rpm for 1min, and discard the waste liquid. Add about 500μL HBCBuffer to the same adsorption column, centrifuge at 12000rpm for 1min, and discard the waste liquid. Add about 700μL DNA Wash Buffer to the same adsorption column, centrifuge at 12000rpm for 1min, and discard the waste liquid. Repeat adding 700μL DNA Wash Buffer, centrifuge at 12000rpm for 1min, and discard the waste liquid. Incubate the adsorption column at 12000rpm for 2min. Leave it uncapped to evaporate the ethanol. Transfer the adsorption column to a new 1.5mL centrifuge tube. Add 35μL sterile ddH2O (deionized water) to the center of the column membrane, cover it and let it stand for 1min, centrifuge at 12000rpm for 1min, and store it at 4℃. Use an ultra-micro spectrophotometer to measure the concentration of the extracted plasmid.

[0040] 4. Identification of recombinant plasmid

[0041] (1) Recombinant plasmid digestion identification:

[0042] The extracted CCHFV-N gene plasmid was double-digested with restriction endonucleases EcoRⅠ and XhoⅠ. The digestion products were subjected to agarose gel electrophoresis. The bottom band was the CCHFV-N gene fragment. The fragment size was consistent with the expectation and was digested into two fragments of 684 bp and 923 bp. The results are as follows: Figure 1 shown.

[0043] (2) Identification of recombinant plasmid protein expression

[0044] The synthesized HeV-N gene plasmid was transfected into 293T cells under the culture conditions of DMEM+10% FBS+1% P / S, 5% carbon dioxide (CO2), and 37°C. After 24 hours, the cell protein samples were collected. The expression of HeV-N protein was verified by protein immunoblotting. The HeV-N protein was expressed correctly. The results are as follows Figure 2 shown.

[0045] Example 3: Preparation of HeV-N gene recombinant plasmid standard

[0046] According to the recombinant plasmid size and plasmid concentration, the copy number of the plasmid in the plasmid stock solution was calculated according to the following formula:

[0047]

[0048] Y represents the copy number, and X represents the measured plasmid concentration in ng / μL.

[0049] The HeV-N gene plasmid size was 6993 bp and the concentration was 711.2 ng / μL. The calculated copy number was 9.276×10 10 copies / μL.

[0050] Dilute the HeV target gene recombinant plasmid stock solution with TE Buffer to a concentration 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 The standard samples were divided into 1000 copies / μL and stored in a -80℃ refrigerator for future use.

[0051] Example 4: Construction and optimization of TaqMan qPCR detection method

[0052] The concentration was 1×10 7The HeV target gene plasmid standard with 100 copies / μL was used as a template, and Premix ExTaqTM (Probe qPCR) was used to construct a single-plex TaqMan qPCR detection method. Three replicate wells were set for the HeV target gene plasmid standard, and three negative controls were set. The reaction system was prepared as follows: Premix Ex Taq (Probe qPCR) (2X) 10μL; forward primer (10μM) 0.4μL; reverse primer (10μM) 0.40μL; Probe probe (10μM) 0.8μL; DNA template 2.0μL; ddH2O 6.4μL; the total system was 20μL. The mixed solution was added to a 96-well plate, sealed, and reacted on the LightCycler 480 System. The qPCR reaction conditions were as follows: 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.

[0053] Based on the established qPCR method, a concentration of 1×10 7 The HeV target gene plasmid standard with 100 copies / μL was used as the template, the amount of probe was fixed, and the amount of upstream and downstream primers with a concentration of 10μM was 0.5, 1.0, 1.5, 2.0, and 2.5μL respectively; the amount of primer was fixed, and the amount of probe with a concentration of 10μM was 0.5, 1.0, 1.5, 2.0, and 2.5μL; negative control and blank control were set up, and the blank group was replaced with sterile ddH2O. The test was carried out in parallel for 3 times, and the Ct value and fluorescence intensity were used as the judgment criteria to determine the optimal primer and probe concentration.

[0054] Based on the established qPCR method, a 1×10 7 The HeV target gene plasmid standard with 100 copies / μL was used as a template, and the optimal combination of primers and probes was used to optimize the annealing temperature of the TaqMan qPCR detection method. The temperature range was set to 58-66°C, i.e. 58, 60, 62, 64, and 66°C. Negative controls and blank controls were set for each temperature gradient. The optimal annealing temperature was determined by comparing the Ct value, fluorescence intensity, and whether nonspecific amplification occurred.

[0055] The experiment showed that the optimal annealing temperature of the HeV-N reaction system after optimization was 60°C. The optimal forward and reverse primer concentrations were 0.1 μM, and the probe concentration was 0.1 μM (Ct value = 17.84 ± 0.1). The results are shown in Table 2.

[0056] Table 2 Ct values ​​of single-plex qPCR at different probe and primer concentrations for HeV-N

[0057]

[0058] Example 5: Establishment of TaqMan qPCR standard curve

[0059] Dilute the HeV-N gene plasmid standards of different concentrations with TE Buffer to 1.0×10 10 copies / μL, and then make a 10-fold gradient dilution to prepare a concentration of 10 0 -10 10 The above standards were used as templates, and Premix Ex TaqTM (ProbeqPCR) was used under the optimal qPCR reaction conditions determined in the early stage to perform single-plex qPCR detection and draw a standard curve. The HeV-N standard curve is obtained from the figure: Y = -3.2585X + 38.738; R 2 =0.999, indicating that there is a good correlation between the logarithmic value of the template amount at different concentration gradients and the Ct value; the amplification efficiency E = 102.72%, indicating that the amplification efficiency of the qPCR method is good. The HeV-N standard curve is shown in Figure 3 shown.

[0060] Example 6: Single-plex qPCR sensitivity test

[0061] The concentration of the aliquot is 10 10 -10 0 The HeV target gene plasmid standard with 10 copies / μL was used as a template, and the qPCR test was performed under the reaction conditions determined in the early stage. The sensitivity of the method was tested with a Ct value of ≤35 as the threshold. The minimum copy number of the HeV-N gene that could be detected was 10 copies / μL. It was concluded that its sensitivity was high. Figure 4 shown.

[0062] Example 7: Single-plex qPCR specificity assay

[0063] Based on the preliminary evaluation of primer and probe specificity by NCBI Blast, this method was used to detect Nipah virus (NiV-N), Rift Valley fever virus (RVFV-N), Hendra virus (HEV-N) gene plasmids, and other viruses preserved in this laboratory, including avian influenza virus (AIV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), Sendai virus (SeV) and other virus N gene positive nucleic acids were selected as samples to be tested. At the same time, negative (sterile water) controls were set up, and qPCR detection was performed using the established detection method to evaluate the specificity of the method. The results showed that the results of other virus gene positive nucleic acids were all negative, and only the HeV-N gene plasmid group showed an amplification curve, indicating that this method has high specificity, such as Figure 5 shown.

[0064] Example 8: Single-plex qPCR repeatability test

[0065] Different concentrations of HeV target gene plasmid standards were used as templates, with a concentration of 10 10 -10 2 The qPCR reaction was performed in parallel with 9 different concentration gradients of 100 copies / μL, and the Ct value of each group was recorded as an intra-batch repeat test; three days later, the same batch of standards frozen in a -80℃ refrigerator were used again for qPCR reaction, and the Ct value of each group was recorded as an inter-batch repeat experiment. The mean (M), standard deviation (SD) and coefficient of variation (CV) of the nucleic acid amplification Ct values ​​of standards with different concentrations were calculated respectively, and the repeatability of the method was evaluated based on the results of two repeated tests. The results are shown in Table 3.

[0066] Table 3 Ct values ​​of HeV-N qPCR parallel experiments

[0067]

[0068] Note: Copies, copy number; Average, average; SD = standard deviation, standard deviation; CV = Coefficient of Variation, coefficient of variation.

[0069] The results in Table 3 show that the coefficient of variation is less than 5%, indicating that the method has good repeatability and stable results.

Claims

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

3.

2. A Hendra virus Taqman probe fluorescent quantitative PCR detection kit as claimed in claim 1, characterized in that: The kit also includes: Premix Ex Taq (Probe qPCR) (2X), Hendra virus DNA template and sterile water.

3. A Hendra virus Taqman probe fluorescent quantitative PCR detection kit as claimed in claim 2, characterized in that: The kit contains 0.4 μL each of Hendra virus forward and reverse primers, 0.8 μL of Taqman probe, 10 μL of Premix Ex Taq (Probe qPCR) (2X), 2 μL of Hendra virus DNA template, and 6.4 μL of sterile water.

4. A Hendra virus Taqman probe fluorescent quantitative PCR detection kit as claimed in claim 3, characterized in that: The optimal forward and reverse primer concentrations in the kit are 0.1 μM, and the Taqman probe concentration is 0.1 μM.

5. A Hendra virus Taqman probe fluorescent quantitative PCR detection kit as claimed in claim 3, characterized in that: 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 60°C for 30 s, 45 cycles; melting curve analysis: 50°C for 30 s, 1 cycle; the reaction is complete.

6. A Hendra virus Taqman probe fluorescent quantitative PCR detection kit as claimed in claim 4, characterized in that: The minimum copy number of Hendra virus that can be detected by the kit is 10 copies / μL.

7. Use of a Hendra virus Taqman probe fluorescent quantitative PCR detection kit as described in any one of claims 1 to 6 in clinical nucleic acid sample detection.

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