CN117487966A - Zika virus nucleic acid detection kit and detection method - Google Patents
Zika virus nucleic acid detection kit and detection method Download PDFInfo
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Abstract
The invention discloses a Zika virus nucleic acid detection kit and a detection method, wherein the detection kit comprises Pfago protein, gDNAs with sequences shown in SEQ ID NO. 1-3 and molecular beacons with sequences shown in SEQ ID NO. 4. The invention establishes a novel nucleic acid detection system based on PfAgo-mediated nucleic acid detection, and targets the nonstructural protein 5 region of the Zika virus genome. In the case of PCR pre-amplification, the MDC of the method of the invention is about 10nM; after the amplification step is introduced, MDC can be greatly reduced to 8.3aM; in addition, the diagnostic results of the ZIKV-PAND clinical simulation samples show 100% consistency with qRT-PCR detection, which would be helpful for clinical diagnosis of ZIKV infection and molecular detection of epidemiological investigation.
Description
Technical Field
The invention belongs to the technical field of Zika virus detection, and particularly relates to a Zika virus nucleic acid detection kit and a detection method.
Background
ZiKV virus (ZIKV) belongs to the Flaviviridae, and is a small envelope positive strand RNA virus composed of a single-stranded 11kb RNA genome. The multimeric proteins encoded by their genomes are divided into three structural proteins (envelope protein (C), pre-membrane protein (prM) and envelope protein (E)) and seven non-structural proteins (NS 1, NS2A, NS2B, NS, NS4A, NS4B and NS 5). As a mosquito-borne flavivirus, the virus is transmitted mainly by Aedes aegypti, but also by sexual intercourse, blood transfusion and mother and infant. Zika virus infection is often asymptomatic or slightly symptomatic, but is rare in adults and children to cause more severe clinical diseases such as Guillain-Barre syndrome. In addition, infection with Zika virus during gestation can cause Congenital Zika Syndrome (CZS), including microcephaly and other congenital deformities, and the like, resulting in fatal death in utero. At present, no specific antiviral drugs and vaccines for treating and preventing the Zika virus infection exist, so that early detection and accurate diagnosis of the Zika virus are important for controlling the transmission of the Zika virus and the influence on public health.
For the detection of Zika virus, isolation of the virus from cell culture is currently considered to be a "gold standard". More serological detection methods, such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA), plaque reduction neutralization assay (PRNT), immunofluorescence assay (IFA), reporter virus neutralization assay (RVNT), multiplex Microsphere Immunoassays (MIA) have been developed for the zika virus detection. Zika virus antibodies may be conjugated to other homologous flaviviruses such as dengue virus (DENV) [12] Cross-reactions were generated (Zhang, x.et al, med Res Rev 2021,41,2039-2108) and therefore further improvements in serologic analysis were needed to address sensitivity and specificity limitation issues.
The molecular detection methods such as RT-PCR, qRT-PCR, yellow fever virus RT-PCR, nested RT-PCR, liquid drop digital PCR (ddPCR) and the like have extremely high sensitivity and specificity, thereby playing an important role in the detection and verification of the Zika virus. The CRISPR/Cas system is used as an accurate and rapid biosensor, and provides a new diagnosis method for detecting infectious diseases including virus nucleic acid detection. For the detection of the zika virus, a Cas 13-based shaerlock (specific high sensitivity enzymatic report unlocking) platform can detect and distinguish between serotypes of the zika virus and four dengue viruses in patient samples, at concentrations as low as1 copy per microliter. However, CRISPR/Cas systems rely on in situ hybridization motifs (PAMs) and the cost of synthesis of the required guide RNAs (grnas) is high.
Disclosure of Invention
In view of this, the present invention aims to create a novel Zika virus nucleic acid detection system, specifically based on PyrococcusfuriosusArgonaute (PfAgo) mediated nucleic acid detection (PAND) technology.
The technical scheme of the invention is as follows:
the first aspect of the invention provides a PfAgo-based Zika virus detection kit, which at least comprises:
pfago protein;
gDNAs having sequences shown in SEQ ID No.1 to 3;
a molecular beacon with a sequence shown as SEQ ID NO. 4.
Preferably, in the above detection kit, both ends of the molecular beacon are respectively provided with a fluorescent group and a quenching group. In one embodiment of the invention, the 5 'end of the molecular beacon is provided with a fluorescent group FAM, and the 3' end of the molecular beacon is provided with a quenching light group BHQ1.
In the detection kit provided by the invention, 3 gDNAs are three guide DNAs designed for the nonstructural protein 5 (ZIKV NS 5) region of the Zika virus genome, and are respectively marked as gr, gt and gf; after phosphorylating gDNAs, three 5'P-gDNAs could direct PfAgo to cleave the Zika virus target DNA (ZIKV target DNA), yielding 16nt of 5' -phosphorylated ssDNA (designated 5'p-gn); 5'p-gn is further used as a second round gDNA to combine with the apo form of Pfago, and the complementary molecular beacon is cleaved, so that the fluorescent group and the quenching group in the molecular beacon are separated to generate fluorescent signals, and the ZIKV can be qualitatively and quantitatively analyzed through fluorescent detection.
Preferably, the above detection kit further comprises Polymerase Chain Reaction (PCR) amplification reagents for specifically amplifying the zhai card virus NS5, and the detection sensitivity can be significantly improved by enriching the target region by pre-amplification using these amplification reagents. "Pre-amplification" as used herein includes, but is not limited to, conventional PCR, isothermal amplification techniques, and the like. As under the detection conditions of one embodiment of the present invention, the Minimum Detection Concentration (MDC) was 10nM (10 fmol/. Mu.L) without pre-amplification, whereas target concentrations as low as 8.3aM (5.0 copies/. Mu.L) could be detected after PCR pre-amplification.
More preferably, in the above detection kit, the PCR amplification reagent comprises primers with sequences shown as SEQ ID NO.10-11, and the target with the sequence shown as SEQ ID NO.12 can be obtained by RT-PCR by using the primer pair.
Based on the detection kit, the second aspect of the invention provides a method for detecting Zika virus, which specifically comprises the following steps: 5 'phosphorylation is carried out on the gDNAs, the sample to be detected, the 5' phosphorylated gDNAs, the molecular beacon and the Pfago protein are added into a reaction buffer solution for reaction, and after the reaction is finished, a cleavage product or a fluorescent signal is detected.
Preferably, in the above detection method, the reaction buffer is composed of HEPES, naCl and MnCl 2 Composition is prepared. Wherein the buffer composition is 20mM HEPES (pH 7.5), 250mM NaCl and 0.5mM MnCl 2 And is most preferred.
Preferably, in the above detection method, the reaction conditions are: incubating at 90-98 ℃ for 20-30min; more preferably, the temperature of incubation is 95 ℃.
Preferably, in the above detection method, the sample to be detected is subjected to PCR pre-amplification using the primers shown in SEQ ID NOS.10 to 11 before performing the specific cleavage reaction.
Preferably, in the above detection method, the gDNAs are subjected to 5' phosphorylation using T4 polynucleotide kinase.
Compared with the prior art, the invention has the beneficial effects that:
the invention develops a novel nucleic acid detection system (ZIKV-PAND) aiming at the genome NS5 of the Zika virus based on Pfago protein, and provides a novel detection tool for rapid and accurate diagnosis of the Zika virus.
The invention can detect the target concentration as low as 8.3aM (5.0 copies/. Mu.L) by the combined method of ZIKV-PAND and PCR pre-amplification, and compared with qPCR detection, the ZIKV-PAND does not need complex laboratory equipment or experienced operators, so that the detection cost and complexity can be reduced. The experimental result of the invention also shows that gDNA in ZIKV-PAND is only suitable for detecting Zika virus, and does not cross react with DENV1 genome, which means that ZIKV-PAND has higher nucleic acid diagnosis specificity. Since PfAgo can distinguish between different nucleotide mutants, multiplex detection of arboviruses such as ZIKV, DENV and chikungunya in single tube reactions will be validated in future clinical diagnostics. In addition, the clinical simulated sample diagnostic results of ZIKV-PAND showed 100% agreement with the qRT-PCR test results, which may be helpful for clinical diagnosis of zika virus infection and molecular detection of epidemiological investigation.
Drawings
FIG. 1 is a schematic diagram of the workflow of the Zika virus nucleic acid detection system provided by the invention;
FIG. 2 is a diagram showing SDS-PAGE analysis result of His-Pfago recombinant proteins used in the present invention under different purification conditions;
FIG. 3 is a graph showing the identification results of guide DNA and molecular beacons in the Pfago-mediated Zika virus nucleic acid detection system of example 1, wherein 28nt MB-ZIKV/f-MB-ZIKV is a cleavage target, 16nt gn/gn1/gn2 is gDNA, and CP is a cleavage product;
FIG. 4 is a schematic diagram of the operation of a nucleic acid detection system based on three gDNA mediated Zika viruses of example 1;
FIG. 5 is a graph showing the results of detection of Zika virus nucleic acid in example 1;
FIG. 6 is a graph showing the result of sensitivity analysis of the Zika virus nucleic acid detection system provided by the invention;
FIG. 7 is a diagram showing the result of target sequence alignment of Zika virus and dengue virus in the present invention;
FIG. 8 is a diagram of the result of specific analysis of the Zika virus nucleic acid detection system provided by the invention;
FIG. 9 is a graph showing the results of a simulation analysis of a sample using the Zika virus nucleic acid detection system provided by the present invention;
in the figure, negative and positive controls are represented by minus and plus signs; the data involved were collected on three independent experiments, expressed as mean ± SD, where n=4 replicates.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following examples. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The invention establishes a ZiKV-PAND nucleic acid detection system based on Pfago, which can guide Pfago to cut Zika virus target DNA through 3 5'p-gDNAs to generate 5' phosphorylated ssDNA, and the 5' phosphorylated ssDNA can be used as new guide DNA to guide Pfago to specifically cut a molecular beacon so as to generate a detectable fluorescent signal, and the work flow of the detection system is shown in figure 1.
The Pfago used in the examples below is in particular a His-Pfago recombinant protein expressed in E.coli with gDNA endonuclease activity, which can be prepared and purified in the manner described in reference (Novel Nucleic Acid Detection for Human Parvovirus B19 Based on PyrococcusfuriosusArgonaute protein viruses 2023,15, doi:10.3390/v 15030595), wherein the results of the determination of His-Pfago recombinant protein affinity-purified with Ni-NTA are shown in FIG. 2.
Primers, gDNA, ssDNA, etc. used in the examples below were synthesized by Shanghai Sanguang Biotechnology Co., ltd. Using T4 polynucleotide kinase from New England Biolabs (MA, USA); 2X HieffTM PCR Master Mix and Hieff-qPCR SYBR Green Master Mix for PCR and qPCR reactions were purchased from Yeason Biotech (Shanghai, china), hiScriptII Q RT SuperMix from Vazyme (Shanghai, china). Other reagents and materials used in the examples below were obtained commercially unless otherwise specified.
The methods used in the examples below are conventional, unless otherwise specified.
EXAMPLE 1 Pfago-mediated establishment of ZIKV nucleic acid detection System
gDNA (gr, gt, gf) for the ZIKV NS5 conserved region and gMB-ZIKV from which molecular beacons can be obtained were first designed, and non-fluorescent MB-ZIKV was designed according to gMB-ZIKV. Different gMB-ZIKV (phosphorylation) and their corresponding MB-ZIKV were synthesized artificially, specifically cleaved and identified with Pfago protein (incubation at 95℃for 20 min), and the identification results were displayed on 20% TBE-PAGE electrophoresis. In which part of the results are shown in FIG. 3A, gn+MB has a very good specific cleavage effect relative to other combinations. It should be noted that the prior art shows that PfAgo has different specific cleavage effects on different target sequences, but the reasons and specific preference rules are still not clear at present.
Further, a fluorescent group (FAM) and a quenching group (BHQ 1) were attached to both ends of MB to obtain a molecular beacon f-MB-ZIKV, and the result of Pfago-specific cleavage of gn-mediated f-MB-ZIKV was shown by 20% TBE-PAGE under white light (FIG. 3B, 1-2) and blue light (FIG. 3B, 3-4), respectively, or imaged in a tube using a blue light transilluminator (FIG. 3C, 1-2). The sequence information of gMB-ZIKV and its corresponding MB-ZIKV shown in FIG. 3 is specifically shown in Table 1.
TABLE 1
From the above detection results, gn+mb has excellent specific cleavage effect under PfAgo mediation, and 3 gdnas capable of guiding PfAgo to cleave ZIKV target DNA to obtain gn are specifically shown as follows:
gt:AGCCAATTGATGATAG(SEQ ID NO.1)；
gf:GTTTGCACATGCCCTC(SEQ ID NO.2)；
gr:ACCTGAGGGCATGTGC(SEQ ID NO.3)。
the above mentioned gt, gr, gf are phosphorylated by T4 polynucleotide kinase (T4 PNK), and the sequence shown in SEQ ID NO.12 is used as the target DNA of Zika virus (recorded as ZIKV NS5-p,249bp, and can be obtained by PCR amplification by using the ZIKV-NS5-F/R with the sequence shown in SEQ ID NO. 10-11) to construct a detection reaction system, specifically: 2pmol of 5' g-DNA, 0.5pmol of ZIKV NS5-P, 0.5pmol of f-MB-ZIKV, and optionally His-Pfago recombinant protein (45 pmol) were reacted in a total volume of 20. Mu.L of Pfago reaction buffer (20 mM HEPES pH7.5, 250mM NaCl and 0.5mM MnCl 2 ) Incubated at 95℃for 20 min. After the reaction was completed, the cleavage products were analyzed by 20% TBE-PAGE electrophoresis, stained with SYBRgold nucleic acid dye, or the fluorescence intensity was measured using a blue light transmission fluorometer and microplate reader. The working principle of the detection system is shown in figure 4.
FIG. 5A is a photograph of a TBE-PAGE analysis of ZIKV-PAND detection system with three guides, f-MB-ZIKV CP and ZIKV NS5-pp, as cleavage products, stained with SYBR Gold dye (lanes 1-2) or directly with UV light recorded fluorescent images (lanes 3-4). FIG. 5B shows the fluorescence intensity results of the three phosphorylated gr/gf/gt-based ZIKV-PAND detection system described above, wherein PfAgo is not added to N.
As can be seen from FIG. 5, after ZIKV-PAND reaction, 249bp of Zika virus target DNA was cleaved to generate 16nt of 5'P-gn, thereby initiating the second round of cleavage of fluorescent molecular beacon (f-MB-ZIKV). Furthermore, the fluorescence intensity detection results showed that PfAgo had 34-fold higher cleavage activity than the control (left panel in fig. 5B); the apparent difference in fluorescence intensity can also be measured using a blue light transmission fluorometer (right panel in fig. 5B). These results indicate that ZIKV-PAND is established and can be used for the detection of Zika virus.
Example 2 sensitivity analysis of ZIKV-PAND detection
To evaluate MDC and sensitivity of ZIKV-PAND, the present example performed PCR pre-amplification or no PCR pre-amplification on ZIKV-PAND.
For ZIKV-PAND sensitive detection without PCR, the sequence amounts of the ZIKV NS5 target PCR products were adjusted to final concentrations of 50nM, 30nM, 25nM, 20nM, 15nM, 10nM, 5nM, 3nM, 1nM or 0.5nM, and ZIKV-PAND detection was then performed with reference to example 1.
In the ZIKV-PAND sensitivity detection by PCR, the initial concentration of the Zika virus plasmid (pUC-ZIKV, containing the sequence shown in SEQ ID NO. 12) was determined to be 6.73X10 8 aM(4.05×10 8 mu.L) and then subjected to 10-fold gradient dilution to a final concentration of 6.73X10 8 aM to 0.67aM (0.41 copies/. Mu.L). Then, the ZIKV NS5 target PCR product was obtained by PCR amplification using 1. Mu.L of the designated ZIKV plasmid as a template and using NS-F/R in a total reaction volume of 10. Mu.L. Finally, a ZIKV-PAND test was performed with reference to example 1.
The results of the test are shown in FIG. 6, wherein the left graph in FIG. 6 shows the MDC analysis results of ZIKV-PAND mediated by three gDNA without PCR, and the right graph in FIG. 6 shows the MDC analysis results of ZIKV-PAND mediated by three gDNA with PCR. As can be seen from FIG. 6, the MDC of ZIKV-PAND without pre-amplification was 10nM (10 fmol/. Mu.L), and when PCR pre-amplification was performed, ZIKV-PAND could detect target concentrations as low as 8.3aM (5.0 copies/. Mu.L).
As a control, qRT-PCR sensitivity detection was performed using ZIKV-qPCR-F/R primers with sequences shown in SEQ ID No. 13-14, wherein the reaction procedure was: pre-denaturation at 95 ℃ for 5min;95 ℃, 10s,60 ℃, 30s,40 cycles; then, the mixture was extended at 60℃for 1min. The detection results showed that qRT-PCR can detect target concentrations as low as 1.67aM (1.0 copies/. Mu.L).
It can be seen that the sensitivity of the method of the invention is slightly lower than qRT-PCR after the introduction of the amplification step; however, compared to qRT-PCR detection, ZIKV-PAND does not require complex laboratory equipment or experienced operators, which can reduce the cost and complexity of the detection.
Example 3 specificity analysis of ZIKV-PAND detection
The Zika virus and dengue virus have the same aedes (Aedes aegypti) vector and geographical distribution, but these two viruses are not clinically distinguishable. Therefore, in ZIKV nucleic acid diagnosis, cross contamination is a common problem in susceptibility detection methods such as qRT-PCR and RT-LAMP.
To demonstrate the specificity of the ZIKV-PAND assay provided by the invention, the sequence with highest similarity to the ZIKV NS5 target in the DENV genome is determined by comparing the gene sequences of ZIKV and DENV (see FIG. 7, specifically shown as SEQ ID NO. 15), and then the sequence is cloned into pUC-18 to obtain dengue virus plasmid (pUC-DENV), and the dengue virus plasmid is used as a target for subsequent PAND assay according to example 2.
Referring to examples 1 and 2, PAND detection was performed using pUC-ZIKV and pUC-DENV, respectively or in combination, and the detection results are shown in fig. 8: when the DENV is used for replacing the ZIKV NS5 target, cross contamination can not be observed in detection; in addition, when ZIKV and DENV were used in combination, no significant effect on the detection of the zika virus targets was observed. These results indicate that the combination of ZIKV-PAND and PCR has very high specificity, and is beneficial to clinical diagnosis of ZIKV infection.
Example 4 sample simulation analysis of ZIKV-PAND
As ZiKV virus is not RNA, the ZIKV nucleic acid detection is carried out on clinical simulation samples by respectively utilizing a ZIKV-PAND system and qRT-PCR, and the specific method is as follows: constructs ZIKV-EGFP (i.e., containing the target sequence shown in SEQ ID NO. 12) and DENV-EGFP (containing the target sequence shown in SEQ ID NO. 15), driven by the CMV promoter, were first constructed separately and transfected into 293 cells, and EGFP expression was observed (FIG. 9A), which means that the target ZIKV and DENV RNAs had been transcribed, and were used as mock samples; after RNA was extracted from the total cells, cDNA was synthesized and used for subsequent ZIKV-PAND and qRT-PCR analysis (see example 2).
The results show that: samples containing ZIKV target RNA but no DENV RNA could be detected using ZIKV-PAND binding PCR (fig. 9B), and the results of qRT-PCR detection and ZIKV-PAND detection were also 100% identical (fig. 9C).
In summary, the invention establishes a novel nucleic acid detection system for Zika virus, which targets the non-structural protein 5 region of the Zika virus genome based on PfAgo-mediated nucleic acid detection. The minimum detection concentration of the detection system is about 10nM under the condition of no PCR pre-amplification, and MDC can be greatly reduced to 8.3aM after the amplification step is introduced; in addition, the diagnosis result of the ZIKV-PAND on the clinical simulation sample shows 100% of consistency with the qRT-PCR detection method.
The foregoing description of the preferred embodiments of the present invention should not be taken as limiting the scope of the invention, and it should be noted that any modifications, equivalents, improvements and others within the spirit and principles of the present invention will become apparent to those of ordinary skill in the art.
Claims (10)
1. The Zika virus detection kit based on Pfago is characterized by comprising:
pfago protein;
gDNAs having sequences shown in SEQ ID No.1 to 3;
a molecular beacon with a sequence shown as SEQ ID NO. 4.
2. The PfAgo-based zika virus detection system of claim 1, wherein said detection kit further comprises an amplification reagent for specifically amplifying zika virus NS 5.
3. The PfAgo-based village card virus detection system according to claim 1, wherein said amplification reagent comprises a primer having a sequence shown in SEQ ID No. 10-11.
4. The PfAgo-based village card virus detection system of claim 1, wherein said molecular beacon has a fluorescent group and a quencher group.
5. The PfAgo-based method for detecting the Zhai Ka virus is characterized in that the method is not used for diagnosing and treating diseases, and is carried out by adopting the detection kit as set forth in any one of claims 1 to 4, and specifically comprises the following steps: 5 'phosphorylation is carried out on the gDNAs, the sample to be detected, the 5' phosphorylated gDNAs, the molecular beacon and the Pfago protein are added into a reaction buffer solution for reaction, and after the reaction is finished, a cleavage product or a fluorescent signal is detected.
6. Root of Chinese characterThe method for detecting Pfago-based Zika virus according to claim 5, wherein said reaction buffer consists of HEPES, naCl and MnCl 2 Composition is prepared.
7. The PfAgo-based method for detecting a card virus according to claim 5, wherein the reaction conditions are: incubating at 90-98 ℃ for 20-30min.
8. The PfAgo-based method of claim 7, wherein said incubation is at a temperature of 95 ℃.
9. The PfAgo-based method for detecting a Zika virus according to claim 5, wherein the primers shown in SEQ ID NOS.10 to 11 are used for PCR pre-amplification of the sample to be detected.
10. The PfAgo-based village card virus detection method according to claim 5, wherein said gDNAs are 5' phosphorylated using T4 polynucleotide kinase.
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