A Nipah virus Taqman probe fluorescent quantitative PCR detection kit and its application

Abstract

The invention discloses a Nipah virus Taqman probe fluorescent quantitative PCR detection kit and its application, by designing specific primers and corresponding fluorescent probes of Nipah virus, then selecting the conserved gene sequence of the virus using the known viral genome sequence, constructing a standard recombinant plasmid containing the conserved gene sequence using gene cloning technology, then after identifying the recombinant plasmid, preparing a plasmid standard, and then obtaining a standard curve, and evaluating the sensitivity, specificity and repeatability of the PCR method, and finally forming a detection kit with a fluorescent quantitative PCR reaction system. The kit detection operation is simple and efficient, can realize the rapid detection of Nipah virus, and has the characteristics of high sensitivity, strong specificity and good repeatability, and can be widely used in clinical nucleic acid sample detection.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and relates to a Nipah virus Taqman probe fluorescent quantitative PCR detection kit and application thereof. Background Art

[0002] Nipah virus (NiV) is a highly pathogenic zoonotic virus of the Paramyxoviridae family. It is an RNA virus and is classified as a biosafety level 4 virus. Nipah virus was first discovered in a pig farm in Sungai Nipah, Negeri Sembilan, Malaysia in 1997, hence the name. Its natural host is bats (especially fruit bats), and intermediate hosts include susceptible animals such as pigs, horses, cats, and dogs. At the same time, NiV has the ability to spread from person to person. After human infection, it usually manifests as symptoms of acute encephalitis and respiratory diseases, with a high mortality rate. The main transmission routes of NiV include respiratory transmission (such as droplet transmission), contact transmission (such as contact with secretions and excretions of patients or infected animals, etc.), and transmission through contaminated medical devices (such as syringes, etc.). From October 1998 to May 1999, NiV broke out in Malaysia on a large scale, causing 265 people to become ill and 105 people to die. In May 2018, NiV was prevalent in Kerala, southern India, killing at least 11 people. In 2023, NiV occurred in Bangladesh, with 11 confirmed cases and 8 deaths reported; 6 cases were confirmed in India, including 2 deaths. So far, no cases of NiV infection have been found in my country, but my country is geographically close to Southeast Asia and South Asia where NiV is frequent, and tourism and economic and trade exchanges are frequent. This geographical proximity increases the risk of NiV being introduced into my country. In addition, fruit bats, the natural host of NiV, exist in some parts of southern my country, and NiV antibodies have been detected in them, which further increases the possibility of NiV spreading in my country. At the same time, its multi-path transmission and high morbidity and mortality characteristics pose a serious threat to public health. In addition, there are currently no effective drugs and vaccines for NiV, which increases the difficulty and urgency of epidemic prevention and control. Therefore, strengthening the detection and research of NiV is of great significance for timely detection of epidemics, control of transmission, and reduction of morbidity and mortality.

[0003] The laboratory diagnostic methods for NiV are mainly divided into serological diagnostic methods and molecular diagnostic methods. Traditional serological diagnostic methods mainly include enzyme-linked immunosorbent assay and serum neutralization test. Molecular diagnostic methods mainly include a series of detection methods for viral nucleic acids, mainly including real-time fluorescence quantitative PCR (qPCR) and nucleic acid sequencing methods. However, the positive detection rate of serum neutralization test is low; the sensitivity and specificity of enzyme-linked immunosorbent assay are slightly lower than those of molecular detection technology, and NiV cannot be detected quickly, sensitively and specifically.

[0004] 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 due to its high efficiency, specificity and sensitivity. At present, there are no reports on the use of Taqman fluorescent quantitative PCR technology for the detection and diagnosis of NiV. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned technical problems existing in the existing NIV detection methods, and to provide a Nipah 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 Nipah 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 Nipah virus Taqman probe fluorescent quantitative PCR detection kit, comprising a specific qPCR detection primer pair for Nipah 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 Nipah 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 further comprises: Premix Ex Taq (ProbeqPCR) (2X), Nipah virus DNA template and sterile water.

[0011] Furthermore, the kit contains 0.4 μL each of Nipah virus forward and reverse primers, 0.8 μL of Taqman probe, 10 μL of Premix Ex Taq (Probe qPCR) (2X), 2 μL of Nipah 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 62°C for 30 s, 45 cycles; melting curve analysis: 50°C, 30 s, 1 cycle; the reaction is complete.

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

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

[0017] Compared with the prior art, the Nipah 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 Nipah 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 Nipah virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of NIV-N gene recombinant enzyme digestion identification;

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

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

[0024] Figure 4 This is the result of NIV-N sensitivity test;

[0025] Figure 5 These are the results of the NIV-N qPCR specificity experiment. 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] The currently published full genes of NiV were searched on NCBI GeneBank, and after downloading, sequence analysis was performed on the full genes using MEGA7.0 software. By analyzing the results and consulting the literature, relatively conservative target genes (N, F, G) were screened out. Then, MEGA7.0 was used to perform sequence alignment on the target genes to find the core conservative regions. Nipahvirus N gene (NiV-N) was selected to perform sequence alignment on the target genes using MEGA7.0 to find the core conservative regions. Using Oligo7 software and referring to relevant literature, the primer sequences and probe sequences of the conservative gene N gene NiV target gene were designed, and their specificity was determined by Blast verification. The primers and probes were synthesized by BGI. The specific primer sequences and the corresponding nucleotide sequences of the Taqman fluorescent probes are shown in Table 1.

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

[0030]

[0031] Example 2: Identification of NiV-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, and invert and culture in a 37℃ incubator for 15 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 15 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 NiV-N gene plasmid was double-digested with restriction endonucleases EcoRⅠ and XhoⅠ, and the digestion products were subjected to agarose gel electrophoresis. The bottom band was the NiV-N gene fragment, and the fragment size was consistent with the expectation, which was digested into three fragments of 410bp, 453bp, and 745bp. The results are as follows: Figure 1 shown.

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

[0044] The synthesized NiV-N gene plasmid was transfected into 293T cells in the culture conditions of DMEM+10% FBS+1% P / S and 5% carbon dioxide (CO 2) , 37℃. After 20h, the cell protein samples were collected. The expression of NiV-N protein was verified by western blotting. The NiV-N protein was expressed correctly. The results are as follows Figure 2 shown.

[0045] Example 3: Preparation of NiV-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 size of the NiV-N gene plasmid was 6993 bp, the concentration was 542.6 ng / μL, and the copy number was calculated to be 7.077×10 10 copies / μL.

[0050] Use TE Buffer to dilute the NiV target gene recombinant plasmid stock solution 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 7 The single-plex TaqMan qPCR detection method was constructed using the NiV target gene plasmid standard with 10 copies / μL as the template and Premix ExTaqTM (Probe qPCR). Three replicate wells were set for the NiV 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 NiV target gene plasmid standard with 100 copies / μL was used as the template, the probe amount was fixed, and the forward and reverse primers with a concentration of 10μM were used in amounts of 0.5, 1.0, 1.5, 2.0, and 2.5μL respectively; the primer amount was fixed, and the probe with a concentration of 10μM was used in amounts of 0.5, 1.0, 1.5, 2.0, and 2.5μL; a negative control and a blank control were set up, and the blank group was replaced with sterile ddH2O. The test was performed 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 NiV 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 NiV-N reaction system after optimization was 62°C. The optimal upstream / downstream primer concentration was 0.1 μM, and the probe concentration was 0.5 μM (Ct value = 11.53 ± 0.11). The results are shown in Table 2.

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

[0057]

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

[0059] The NiV-N gene plasmid standards of different concentrations were diluted 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 NiV-N standard curve is obtained from the figure: Y = -3.276X + 38.92; R 2 =0.9998, 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 = 101.95%, indicating that the amplification efficiency of the qPCR method is good. The NiV-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 NiV 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 lowest copy number of the NiV-N gene that can be detected is 10 copies / μL. Therefore, it is concluded that its sensitivity is 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 Hendra virus (HeV-N), Rift Valley fever virus (RVFV-N), Nipah virus (NIV-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 viral 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 viral gene positive nucleic acids were all negative, and only the NiV-N gene plasmid group showed an amplification curve, indicating that this method has high specificity, such as Figure 5shown.

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

[0065] Different concentrations of NiV 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 NiV-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 7%, indicating that the method has good repeatability and stable results.

Claims

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

3.

2. A Nipah 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), Nipah virus DNA template and sterile water.

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

4. A Nipah virus Taqman probe fluorescent quantitative PCR detection kit as claimed in claim 3, characterized in that: The forward and reverse primer concentrations in the kit are 0.1 μM, and the Taqman probe concentration is 0.5 μM.

5. A Nipah 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 62°C for 30 s, 45 cycles; melting curve analysis: 50°C, 30 s, 1 cycle; the reaction is complete.

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

7. Use of a Nipah 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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