Zika virus nucleic acid detection kit and detection method

Abstract

The present invention discloses a Zika virus nucleic acid detection kit and a detection method, wherein the detection kit comprises a PfAgo protein, gDNAs with sequences as shown in SEQ ID NO.1 to 3, and a molecular beacon with a sequence as shown in SEQ ID NO.4. The present invention establishes a novel nucleic acid detection system, which targets the non-structural protein 5 region of the Zika virus genome based on PfAgo-mediated nucleic acid detection. In the case of PCR pre-amplification, the MDC of the method of the present invention is about 10nM; after the introduction of the amplification step, the MDC can be greatly reduced to 8.3aM; in addition, the diagnostic results of the ZIKV-PAND clinical simulation samples show a consistency of 100% with the qRT-PCR detection, which will be helpful for the clinical diagnosis of ZIKV infection and molecular detection of epidemiological surveys.

Description

Technical Field

[0001] The present invention belongs to the technical field of Zika virus detection, and in particular relates to a Zika virus nucleic acid detection kit and a detection method. Background Art

[0002] Zika virus (ZIKV), belonging to the Flaviviridae family and the genus Flavivirus, is a small, enveloped, positive-strand RNA virus composed of a single-stranded 11kb RNA genome. Its genome encodes a polyprotein composed of three structural proteins (envelope (C), pre-membrane (prM), and envelope (E)) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). As a mosquito-borne flavivirus, the virus is primarily transmitted by Aedes aegypti mosquitoes and can also be transmitted sexually, through blood transfusions, and from mother to child. Zika virus infection is often asymptomatic or mild, but can rarely cause more severe clinical illness, such as Guillain-Barré syndrome, in adults and children. Furthermore, Zika virus infection during pregnancy can cause congenital Zika syndrome (CZS), which includes microcephaly and other congenital malformations, leading to intrauterine fatality. Currently, there are no specific antiviral drugs or vaccines to treat or prevent Zika virus infection. Therefore, early detection and accurate diagnosis of Zika virus are crucial to controlling its spread and its public health impact.

[0003] For the detection of Zika virus, isolation of the virus from cell culture is currently considered the "gold standard". More serological tests, such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA), plaque reduction neutralization test (PRNT), immunofluorescence assay (IFA), reporter virus neutralization test (RVNT), and multiplex microsphere immunoassay (MIA) have been developed for Zika virus detection. Zika virus antibodies may be detected in combination with other homologous flaviviruses such as dengue virus (DENV)

[12] Cross-reactivity occurs (Zhang, X. et al, Med Res Rev 2021, 41, 2039-2108), so further improvement of serological analysis is needed to address the problems of sensitivity and specificity limitations.

[0004] Molecular detection methods such as RT-PCR, qRT-PCR, pan-flavivirus RT-PCR, nested RT-PCR, and droplet digital PCR (ddPCR) have extremely high sensitivity and specificity, and therefore have played an important role in the detection and validation of Zika virus. As an accurate and rapid biosensor, the CRISPR / Cas system has brought new diagnostic methods to infectious disease detection, including viral nucleic acid detection. For the detection of Zika virus, the Cas13-based SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking) platform can detect and distinguish Zika virus and four dengue virus serotypes in patient samples at concentrations as low as 1 copy per microliter. However, the CRISPR / Cas system relies on in situ hybridization motifs (protospacer-adjacent motif, PAM), and the required guide RNA (gRNA) is expensive to synthesize. Summary of the Invention

[0005] In view of this, the present invention aims to establish a novel Zika virus nucleic acid detection system, which is specifically based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection (PAND) technology.

[0006] The technical solutions of the present invention are as follows:

[0007] The first aspect of the present invention provides a Zika virus detection kit based on PfAgo, which comprises at least:

[0008] PfAgo protein;

[0009] gDNAs whose sequences are shown in SEQ ID NOs. 1 to 3;

[0010] The molecular beacon has a sequence as shown in SEQ ID NO.4.

[0011] Preferably, in the above detection kit, the molecular beacon has a fluorescent group and a quenching group at both ends, respectively. In one embodiment of the present invention, the 5' end of the molecular beacon has a fluorescent group FAM, and the 3' end of the molecular beacon has a quenching group BHQ1.

[0012] In the detection kit provided by the present invention, three gDNAs are three guide DNAs designed for the nonstructural protein 5 (ZIKV NS5) region of the Zika virus genome, denoted as gr, gt and gf respectively; after the gDNAs are phosphorylated, the three 5'P-gDNAs can guide PfAgo to cleave the Zika virus target DNA (ZIKV target DNA), generating 16nt 5'-phosphorylated ssDNA (denoted as 5'p-gn); and 5'p-gn then acts as a second round of gDNA to bind to the apo form of PfAgo and cleave the complementary molecular beacon, so that the fluorescent group and quenching group in the molecular beacon are separated to generate a fluorescent signal, and the ZIKV can be qualitatively and quantitatively analyzed by fluorescence detection.

[0013] Preferably, the above-mentioned detection kit also includes polymerase chain reaction (PCR) amplification reagents for specifically amplifying Zika virus NS5. The use of these amplification reagents to enrich the target area through pre-amplification can significantly improve the detection sensitivity. The "pre-amplification" described in the present invention includes but is not limited to conventional PCR, isothermal amplification technology, etc. Under the detection conditions of one embodiment of the present invention, when no pre-amplification is performed, the minimum detection concentration (MDC) is 10nM (10fmol / μL), and after PCR pre-amplification, a target concentration as low as 8.3aM (5.0copies / μL) can be detected.

[0014] More preferably, in the above detection kit, the PCR amplification reagent includes primers with sequences as shown in SEQ ID NOs. 10 to 11, and the target with sequence as shown in SEQ ID NO. 12 can be obtained by RT-PCR using the primer pair.

[0015] Based on the above detection kit, the second aspect of the present invention provides a Zika virus detection method, specifically: gDNAs are 5' phosphorylated, the test sample, 5' phosphorylated gDNAs, molecular beacons and PfAgo protein are added to the reaction buffer for reaction, and the cleavage product or fluorescence signal is detected after the reaction is completed.

[0016] Preferably, in the above detection method, the reaction buffer consists of HEPES, NaCl and MnCl2, wherein the optimal buffer composition is 20mM HEPES (pH 7.5), 250mM NaCl and 0.5mM MnCl2.

[0017] Preferably, in the above detection method, the reaction conditions are: incubation at 90-98°C for 20-30 min; more preferably, the incubation temperature is 95°C.

[0018] Preferably, in the above detection method, before the specific cleavage reaction, the sample to be tested is subjected to PCR pre-amplification using the primers shown in SEQ ID NOs. 10 to 11.

[0019] Preferably, in the above detection method, the gDNAs are 5' phosphorylated using T4 polynucleotide kinase.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] Based on the PfAgo protein, the present invention develops a novel nucleic acid detection system (ZIKV-PAND) targeting the NS5 of the Zika virus genome, providing a new detection tool for the rapid and accurate diagnosis of Zika virus.

[0022] The present invention combines ZIKV-PAND with PCR pre-amplification to detect target concentrations as low as 8.3aM (5.0 copies / μL). Compared to qPCR detection, ZIKV-PAND does not require complex laboratory equipment or experienced operators, which can reduce the cost and complexity of detection. The experimental results of the present invention also show that the gDNA in ZIKV-PAND has been proven to be only suitable for detecting Zika virus and does not cross-react with the DENV1 genome, which means that ZIKV-PAND has higher nucleic acid diagnostic specificity. Because PfAgo can distinguish different nucleotide mutants, multiplex detection of arboviruses such as ZIKV, DENV, and chikungunya fever in a single-tube reaction will be validated in future clinical diagnosis. In addition, the diagnostic results of clinical simulation samples of ZIKV-PAND showed 100% consistency with the qRT-PCR detection results, which may facilitate the clinical diagnosis of Zika virus infection and molecular detection in epidemiological surveys. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic diagram of the workflow of the Zika virus nucleic acid detection system provided by the present invention;

[0024] Figure 2 This is a diagram showing the results of SDS-PAGE analysis of the His-PfAgo recombinant protein used in the present invention under different purification conditions;

[0025] Figure 3 This figure shows the identification results of guide DNA and molecular beacons in the PfAgo-mediated Zika virus nucleic acid detection system in Example 1. In the figure, the 28nt MB-ZIKV / f-MB-ZIKV is the cleavage target, the 16nt gn / gn1 / gn2 is the gDNA, and CP is the cleavage product;

[0026] Figure 4Schematic diagram of the working principle of the three gDNA-mediated Zika virus nucleic acid detection systems in Example 1;

[0027] Figure 5 This is a diagram showing the results of Zika virus nucleic acid detection in Example 1;

[0028] Figure 6 This is a graph showing the sensitivity analysis results of the Zika virus nucleic acid detection system provided by the present invention;

[0029] Figure 7 This is a diagram showing the target region sequence alignment results of Zika virus and dengue virus in the present invention;

[0030] Figure 8 A diagram showing the specificity analysis results of the Zika virus nucleic acid detection system provided by the present invention;

[0031] Figure 9 This is a diagram showing the results of a simulated analysis of a sample using the Zika virus nucleic acid detection system provided by the present invention;

[0032] In the figures, negative and positive controls are indicated by minus and plus signs; the data involved are based on three independent experiments and are expressed as mean ± SD, with n = 4 replicates. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0034] The present invention establishes a Zika virus nucleic acid detection system (ZIKV-PAND) based on PfAgo. The detection system can guide PfAgo to cut Zika virus target DNA through three 5'p-gDNAs, generating 5'phosphorylated ssDNA. The 5'phosphorylated ssDNA can then serve as a new guide DNA to guide PfAgo to specifically cut the molecular beacon, thereby generating a detectable fluorescent signal. The workflow is as follows: Figure 1 shown.

[0035] The PfAgo used in the following examples is specifically a His-PfAgo recombinant protein with gDNA endonuclease activity expressed in Escherichia coli. Its preparation and purification methods can be referred to the literature (Novel Nucleic Acid Detection for Human Parvovirus B19 Based on Pyrococcus furiosus Argonaute Protein. Viruses 2023, 15, doi: 10.3390 / v15030595). The results of the Ni-NTA affinity purification of the His-PfAgo recombinant protein are shown in FIG. Figure 2 shown.

[0036] The primers, gDNA, and ssDNA used in the following examples were synthesized by Shanghai Sanguang Biotechnology Co., Ltd. T4 polynucleotide kinase was purchased from New England Biolabs (MA, USA). 2×Hieff™ PCR Master Mix and Hieff-qPCR SYBR Green Master Mix used in PCR and qPCR reactions were purchased from Yeason Biotech (Shanghai, China), and HiScriptII Q RT SuperMix was purchased from Vazyme (Shanghai, China). All other reagents and materials used in the following examples were commercially available unless otherwise specified.

[0037] The methods used in the following examples are all conventional methods unless otherwise specified.

[0038] Example 1 Establishment of PfAgo-mediated ZIKV nucleic acid detection system

[0039] First, we designed gDNA targeting the conserved region of ZIKV NS5 (gr, gt, gf) and gMB-ZIKV that can obtain molecular beacons. Based on gMB-ZIKV, we designed non-fluorescent MB-ZIKV. Different gMB-ZIKV (phosphorylated) and their corresponding MB-ZIKV were artificially synthesized and identified by specific cleavage using PfAgo protein (incubated at 95°C for 20 minutes). The identification results were displayed on 20% TBE-PAGE electrophoresis. Some of the results are shown in Figure 2. Figure 3 As shown in Figure A, gn+MB has a very good specific cleavage effect compared to other combinations. It should be noted that existing technologies have shown that PfAgo has different specific cleavage effects on different target sequences, but the reasons and specific preference patterns are still unclear.

[0040] Further, the two ends of MB were connected with fluorescent group (FAM) and quenching group (BHQ1) to obtain molecular beacon f-MB-ZIKV. The PfAgo specific cleavage results of f-MB-ZIKV mediated by gn were observed under white light ( Figure 3 B, 1-2), blue light ( Figure 3 B, 3-4) were performed on 20% TBE-PAGE electrophoresis or imaged in tube using a blue light transilluminator ( Figure 3 C, 1-2). Figure 3 The sequence information of gMB-ZIKV and its corresponding MB-ZIKV involved in the experiment is shown in Table 1.

[0041] Table 1

[0042]

[0043]

[0044] From the above test results, it can be seen that gn+MB has an excellent specific cleavage effect under the mediation of PfAgo, and the three gDNAs that can guide PfAgo to cleave ZIKV target DNA to obtain the gn are specifically as follows:

[0045] gt:AGCCAATTGATGATAG(SEQ ID NO.1);

[0046] gf:GTTTGCACATGCCCTC(SEQ ID NO.2);

[0047] gr:ACCTGAGGGCATGTGC (SEQ ID NO. 3).

[0048] The above-mentioned gt, gr, and gf were phosphorylated by T4 polynucleotide kinase (T4 PNK), and the sequence shown in SEQ ID NO.12 was used as the Zika virus target DNA (denoted as ZIKV NS5-p, 249 bp, which can be obtained by PCR amplification using the sequences of ZIKV-NS5-F / R shown in SEQ ID NOs.10-11). The detection reaction system was constructed, specifically: 2 pmol 5'g-DNA, 0.5 pmol ZIKV NS5-P, 0.5 pmol f-MB-ZIKV, with or without His-PfAgo recombinant protein (45 pmol) were incubated in a total volume of 20 μL of PfAgo reaction buffer (20 mM HEPES pH 7.5, 250 mM NaCl and 0.5 mM MnCl2) at 95°C for 20 minutes. After the reaction is complete, the cleavage products are analyzed by 20% TBE-PAGE electrophoresis and then stained with SYBR Gold nucleic acid dye, or the fluorescence intensity is measured using a blue light transmission fluorescence instrument and a microplate reader. Figure 4 .

[0049] Figure 5 A shows the results of TBE-PAGE analysis of the ZIKV-PAND detection system with three guides, stained with SYBR Gold dye (lanes 1-2) or directly recorded with ultraviolet light (lanes 3-4). In the figure, f-MB-ZIKVCP and ZIKV NS5-pp are cleavage products. Figure 5 B is the fluorescence intensity result of the above-mentioned ZIKV-PAND detection system based on three phosphorylated gr / gf / gt. In the figure, N means that PfAgo was not added.

[0050] from Figure 5 It can be seen that after the ZIKV-PAND reaction, the 249bp Zika virus target DNA was cleaved to generate 16nt 5'P-gn, which triggered the second round of cleavage of the fluorescent molecular beacon (f-MB-ZIKV). In addition, the fluorescence intensity detection results showed that the cleavage activity of PfAgo was 34 times that of the control ( Figure 5 B, left); using a blue light transmission fluorescence instrument, the obvious difference in fluorescence intensity can also be measured ( Figure 5 (B, right panel). These results indicate that the ZIKV-PAND has been established and can be used for Zika virus detection.

[0051] Example 2 Sensitivity Analysis of ZIKV-PAND Detection

[0052] To evaluate the MDC and sensitivity of ZIKV-PAND, ZIKV-PAND was detected with or without PCR pre-amplification.

[0053] For sensitive detection of ZIKV-PAND without PCR, the sequence amount of the ZIKV NS5 target PCR product was adjusted to a final concentration of 50 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 3 nM, 1 nM, or 0.5 nM, and then the ZIKV-PAND assay was performed with reference to Example 1.

[0054] When using PCR for ZIKV-PAND sensitivity detection, the initial concentration of the Zika virus plasmid (pUC-ZIKV, containing the sequence shown in SEQ ID NO. 12) was first determined to be 6.73×10 8 aM(4.05×10 8 copies / μL), and then it was diluted 10-fold to a final concentration of 6.73×10 8 The ZIKV NS5 target PCR product was obtained by PCR amplification using NS-F / R in a total reaction volume of 10 μL, using 1 μL of the indicated ZIKV plasmid as template. Finally, the ZIKV-PAND assay was performed as described in Example 1.

[0055] Test results such as Figure 6 As shown, Figure 6 The middle left picture shows the MDC analysis results of three gDNA-mediated ZIKV-PAND without PCR. Figure 6 The middle right picture shows the results of MDC analysis of three gDNA-mediated ZIKV-PAND by PCR. Figure 6 It can be seen that the MDC of ZIKV-PAND without pre-amplification is 10nM (10fmol / μL). When PCR pre-amplification is performed, ZIKV-PAND can detect target concentrations as low as 8.3aM (5.0copies / μL).

[0056] As a control, qRT-PCR sensitivity testing was performed using the ZIKV-qPCR-F / R primers shown in SEQ ID NOs. 13-14. The reaction procedure was as follows: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds, followed by extension at 60°C for 1 minute. The results showed that qRT-PCR could detect target concentrations as low as 1.67 aM (1.0 copies / μL).

[0057] It can be seen that after the introduction of the amplification step, the sensitivity of the method of the present invention is slightly lower than that of qRT-PCR; however, compared with qRT-PCR detection, ZIKV-PAND does not require complex laboratory equipment or experienced operators, which can reduce the cost and complexity of detection.

[0058] Example 3 Specificity Analysis of ZIKV-PAND Detection

[0059] Zika virus and dengue virus share the same Aedes aegypti mosquito vector and geographic distribution, but the two viruses are difficult to distinguish clinically. Therefore, cross-contamination is a common problem in ZIKV nucleic acid diagnosis using susceptible detection methods such as qRT-PCR and RT-LAMP.

[0060] To demonstrate the specificity of the ZIKV-PAND assay provided by the present invention, this example first compared the gene sequences of ZIKV and DENV to determine the sequence in the DENV genome with the highest similarity to the ZIKV NS5 target (see Figure 7 , specifically as shown in SEQ ID NO.15), and then the sequence was cloned into pUC-18 to obtain dengue virus plasmid (pUC-DENV), and it was used as the target for subsequent PAND detection with reference to Example 2.

[0061] Referring to Example 1 and Example 2, pUC-ZIKV and pUC-DENV were used separately or in combination for PAND detection. The test results were as follows: Figure 8 The results show that when the ZIKV NS5 target was replaced by DENV, no cross-contamination was observed; in addition, no significant effect on Zika virus target detection was observed when ZIKV and DENV were mixed. These results indicate that the combination of ZIKV-PAND and PCR has high specificity and is beneficial for the clinical diagnosis of ZIKV infection.

[0062] Example 4: Sample Simulation Analysis of ZIKV-PAND

[0063] Since Zika virus is RNA, this case used the ZIKV-PAND system and qRT-PCR to detect ZIKV nucleic acid in clinical simulation samples. The specific methods were as follows: First, the CMV promoter-driven constructs ZIKV-EGFP (containing the target sequence shown in SEQ ID NO.12) and DENV-EGFP (containing the target sequence shown in SEQ ID NO.15) were constructed and transfected into 293 cells, and EGFP expression was observed ( Figure 9 A), which means that the target ZIKV and DENV RNA have been transcribed and can be used as a mock sample; after extracting the RNA of the total cells, cDNA was synthesized and used for subsequent ZIKV-PAND and qRT-PCR analysis (see Example 2).

[0064] The results showed that ZIKV-PAND combined with PCR could detect samples containing ZIKV target RNA but not DENV RNA ( Figure 9B), and the results of qRT-PCR detection and ZIKV-PAND detection were 100% consistent ( Figure 9 C).

[0065] In summary, the present invention has established a novel nucleic acid detection system for Zika virus. This system, based on PfAgo-mediated nucleic acid detection, targets the nonstructural protein 5 region of the Zika virus genome. Without PCR pre-amplification, this detection system has a minimum detection concentration of approximately 10 nM. With the introduction of an amplification step, the minimum detection concentration (MDC) can be significantly reduced to 8.3 aM. Furthermore, the diagnostic results of ZIKV-PAND on simulated clinical samples showed 100% concordance with those of qRT-PCR detection methods.

[0066] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Zika virus detection kit based on PfAgo, characterized in that: include: PfAgo protein; gDNAs whose sequences are shown in SEQ ID NOs. 1 to 3; A molecular beacon with a sequence as shown in SEQ ID NO.4, wherein the molecular beacon has a fluorescent group and a quenching group; The amplification reagent used for specifically amplifying Zika virus NS5 specifically includes primers whose sequences are shown in SEQ ID NOs.10 to 11.

2. A Zika virus detection method based on PfAgo, characterized in that: The Zika virus detection method is not for the purpose of disease diagnosis and treatment. It is carried out using the detection kit described in claim 1, specifically: gDNAs are 5' phosphorylated, the sample to be tested, 5' phosphorylated gDNAs, molecular beacons and PfAgo protein are added to the reaction buffer for reaction, and the fluorescent signal is detected after the reaction is completed.

3. The Zika virus detection method based on PfAgo according to claim 2, characterized in that: The reaction buffer consisted of HEPES, NaCl and MnCl2.

4. The Zika virus detection method based on PfAgo according to claim 2, characterized in that: The reaction conditions are: incubation at 90-98°C for 20-30 min.

5. The Zika virus detection method based on PfAgo according to claim 4, characterized in that: The incubation temperature is 95°C.

6. The Zika virus detection method based on PfAgo according to claim 2, characterized in that: The primers shown in SEQ ID NOs. 10 to 11 were used to perform PCR pre-amplification on the sample to be tested.

7. The Zika virus detection method based on PfAgo according to claim 2, characterized in that: The gDNAs were 5' phosphorylated using T4 polynucleotide kinase.

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