CN114703328B - Pfago protein mediated B19 virus nucleic acid detection kit and detection method - Google Patents
Pfago protein mediated B19 virus nucleic acid detection kit and detection method Download PDFInfo
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Abstract
The invention belongs to the technical field of B19 virus detection, and discloses a Pfago protein-mediated B19 virus nucleic acid detection kit and a detection method, wherein the detection kit comprises three or single gDNA (global DNA) for guiding Pfago to target the ns1 gene of the B19 virus, and the sequence SEQ ID NO is shown in 1-3; gDNA guides Pfago to specifically cut the ns1 gene fragment of the B19 virus, and further, gn-DNA of a targeting fluorescent molecular beacon is generated to carry out second-round cutting, so that the specificity and sensitivity of the B19 virus are detected. Compared with the existing B19 virus detection method, the method has the advantages of low detection cost, high sensitivity and strong specificity, and has important practical application value.
Description
Technical Field
The invention belongs to the technical field of B19 virus detection, and in particular relates to a method for detecting B19 virusPfAgo protein mediated B19 virus nucleic acid detection kit and detection method.
Background
B19 is one of the only two parvoviruses that have been able to infect and cause human disease to date. As an important pathogen, B19 virus infects different crowds and can cause diseases such as childhood infectious erythema, acute re-obstacle crisis, fetal edema and even stillbirth, and the like, and meanwhile, because the disease can be transmitted through blood transfusion and blood products, B19 virus is closely related to the safety of blood transfusion and the safety of blood products, so that the development of a novel virus detection technology has important application value.
Currently, the B19 virus nucleic acid detection mainly comprises a spot hybridization method, an in situ hybridization method and a polymerase chain reaction method. In recent years, with the advent of novel gene editing technologies typified by CRISPR/Cas9 and the wide application to the detection of pathogenic microorganisms and the like, conventional molecular diagnostic technologies have been put into a novel molecular diagnostic stage with low cost, high sensitivity, and high specificity. As DNA viruses, a novel detection system E-CRISPR method for B19 viruses is also successfully established, mainly based on the property of CRISPR-Cas12a specific cutting DNA, the detection sensitivity reaches pmol level, which shows that the molecular diagnosis method based on the novel gene editing enzyme CRISPR/Cas system has potential application prospect in the B19 virus detection process. However, in view of the expensive synthesis cost of gRNA in CRISPR/Cas systems, it is of great importance to develop new molecular diagnostic methods.
The Argonaute protein is a nucleic acid-guided endonuclease that is involved in RNA silencing or RNA-guided RNA cleavage in eukaryotes; in prokaryotes, however, the proteins cleave ssDNA or ssRNA under the guidance of short DNA sequences, whereNgAgo was once considered a novel gene editing enzyme that replaces Cas 9. Currently, although the method of editing a gene based on boost has not been established successfully, its application in vitro molecular diagnostics has been greatly advanced.PfAgo is a kind of a microorganism which is derived from Gu Shenghuo cocci thermophilusPyrococcus furiosus) The extracted Argonaute protein can cleave ssDNA or ssRNA under the guidance of a short DNA sequence. At the same time, the method comprises the steps of,Pfshort single-stranded DNA generated by Ago cleavage can be used in combination with apo-PfAgo protein binding, initiates a second round of cleavage of the downstream target in the same reaction system, based on which the inventors have establishedPfAgo-mediated nucleic acid detection (PAND) techniques for Human Papilloma Virus (HPV) and SARS-CoV2 nucleic acid detection (He R, wang L, wang F, li W, liu Y, li A, wang Y, mao W, zhai C, ma L. Pyrococcus furiosus Argonaute-mediated nucleic acid detection. Chem Commun (Camb) 2019, 55 (88): 13219-22; wang F, yang J, he R, yu X, chen S, liu Y, wang L, li A, liu L, zhai C, ma L.Pfago-based detection of SARS-CoV-2 Biosens bioelectron 2021, 177:112932）。
Disclosure of Invention
The present invention has for its object to utilizePfThe Ago mediated nucleic acid detection technology realizes the detection of B19 virus and establishes a set of high-sensitivity and specificity B19 virus nucleic acid detection system.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a test kit comprising:
guidancePfAgo protein targeting B19 virusns1The gDNA of the gene is specifically shown as gt-B19 shown in SEQ ID NO. 1, gf-B19 shown in SEQ ID NO. 2 and gr-B19 shown in SEQ ID NO. 3 or gf-B19 shown in SEQ ID NO. 2;
a molecular beacon with a fluorescent group and a quenching group;
andPfAgo protein.
Preferably, the nucleotide sequence of the molecular beacon is shown in SEQ ID NO. 4.
Preferably, the detection kit also comprises a kit for specific amplificationns1Amplification reagents for target sequences on genes; more preferably, the amplification reagents comprise primer pairs as shown in SEQ ID NOS.5-6.
The above-described detection kit can be used for the identification or assisted identification of B19 virus.
Based on the detection kit, the invention establishesPfThe method for detecting the B19 virus nucleic acid mediated by the Ago protein comprises the following specific steps:
s1, amplifying a sample to be detected to obtain tDNA;
s2, setting up gDNA comprising 5' phosphorylation,PfAgo protein, tDNA,PfPerforming a cleavage reaction by using the Ago reaction buffer and a reaction system of the molecular beacon;
and S3, after the reaction is finished, performing fluorescence detection on the reaction system.
Preferably, in the reaction system of step S2, gDNA is reacted withPfThe molar ratio of Ago protein was 1:460.
Preferably, the temperature of the cutting reaction is 90-98 ℃; optimally, the cleavage reaction temperature is 95 ℃.
Preferably, the tDNA content in the reaction system of step S2 is not less than 0.1pmol.
Preferably, the method comprises the steps of,Pfthe Ago reaction buffer included: 200mM HEPES (pH=8.0), 2.5M sodium chloride and 5mM manganese chloride.
The beneficial effects of the invention are as follows:
1) Compared with the guide RNA of the Cas9 system, the guide ssDNA used by the invention not only can be conveniently and stably obtained, but also has the advantage of cost. Meanwhile, through optimizing gDNA in the detection system, the dosage of gDNA in the experimental process is reduced, the process of screening gDNA capable of specifically cutting target DNA is also reduced, and the necessary 5' phosphorylation reaction procedure in the experimental process is further reduced, so that the detection cost is reduced, and the time-consuming and labor-consuming complex process is reduced.
2) The tDNA in the detection system can still be detected after the level of the tDNA is low to pmol, and the detection sensitivity of the tDNA can reach aM after the tDNA is combined with a PCR technology and exceeds the E-CRISPSR method.
Drawings
FIG. 1 shows a His-containing composition prepared according to the present inventionPfConstructing an Ago protein expression vector and an expression purification identification chart;
FIG. 2 shows a His-containing composition prepared according to the present inventionPfA characterization map of the activity of Ago fusion proteins in gDNA mediated cleavage of DNA;
FIG. 3 is a schematic diagram of example 1PfAgo mediated targeting B19 virusns1, a nucleic acid detection method schematic diagram of the gene;
FIG. 4 is a graph of the test results of the detection system constructed in example 1, wherein a is gDNA-B19 guidePfAgo specific cleavage of B19 Virusns1A gel diagram of the gene product, b is an analysis diagram of a fluorescence intensity detection result;
FIG. 5 is a graph showing the sensitivity test results of the test system constructed in example 1;
FIG. 6 is a schematic diagram of example 2PfAgo mediated targeting B19 virusns1, a nucleic acid detection method schematic diagram of the gene;
FIG. 7 is a graph showing the results of the test system constructed in example 2, wherein a is gf-B19 guidePfAgo specific cleavageB19 virusns1A gel diagram of the gene product, b is an analysis diagram of a fluorescence intensity detection result;
FIG. 8 is a graph showing the sensitivity test results of the test system constructed in example 2;
FIG. 9 shows different sets of guide guidesPfGlue pattern of Ago specific cleavage molecular beacons and tDNA.
Detailed Description
The present invention will be described in further detail below for the purpose of making the objects, technical solutions and advantages of the present invention more apparent. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention.
The invention constructsPfAgo mediated targeting B19 virusns1The gene detecting system has first designed target B19 virusns1gDNA of gene, gDNA guidePfAgo specific cleavage of B19 Virusns1The gene fragment further generates gn-DNA of the targeted fluorescent molecular beacon, and the high-sensitivity detection of the B19 virus is realized through the second round of cutting.
PfThe Ago protein may be a natural protein obtained directly from an organism or a recombinant protein. Used in the embodiment of the inventionPfThe Ago protein is recombinant protein, and the preparation process is as follows:
(1) RecombinationPfConstruction of Ago expression vector, induced expression and identification
By means ofpfuDNA polymerase amplificationPfAgo gene (amplification primer isPfAgo-F/R, the sequence of which is shown as SEQ ID NO. 9-10) and reverse amplification pET-23a, gene cloning is carried out by a T5 exonuclease mediated homologous recombination method, and the recombinant plasmid is subjected to the following steps ofNdeI andXhoi after cleavage, the sample was subjected to detection by 0.8% agarose gel. The results are shown in FIG. 1 a (where M is a 1kb DNA marker,1 isXhoI/NdeI enzyme digestion, 2 isXhoI enzyme digestion, 3 isNdeI enzyme digestion, 4 is pET-23a-PfAgo control), the size of the double enzyme digestion product is consistent with the expected result, and preliminary indication of pET-23a-PfThe Ago expression vector was successfully constructed, and then the correct plasmid for enzyme digestion was sent to Shanghai worker for sequencing to further verify the correctness.
pET-23a-PfAgo expression vector transformationE.coliBL21-DE3 strain is induced and expressed for 20 hours by 1.0mM IPTG at low temperature, thalli are collected, ultrasonic bacteria are broken, supernatant is centrifugally taken, a large amount of impurity proteins are removed by water bath at 70-80 ℃, supernatant is centrifugally taken again, filtered by a filter membrane and added into a Ni column affinity chromatography purification column, and the mixture is placed on a mute mixer for incubation for 2 hours at 4 ℃. Sequentially with imidazole containing final concentration of 10mM, 20mM, 50mM, 100mM, 200mMPfAgo lysis buffer (containing 20mM Tris-hcl ph=8.0, 200mM sodium chloride and 0.2mM manganese chloride) was eluted and the protein of interest was collected. Concentration by ultrafiltration dialysis gave a protein concentration of 1.312mg/mL.
In FIG. 1 b is pET-23a-PfAnd (3) performing 8% SDS-PAGE gel electrophoresis of the Ago induced expression product, wherein M is a protein marker,1 is a whole thallus, 2 is a broken bacterial supernatant, 3 is broken bacterial sediment, 4 is a high-temperature treated supernatant, 5 is a flow-through, and 6-10 are sequentially eluted by 10, 20, 50, 100 and 300mM imidazole. The results show that: his after high temperature treatmentPfThe Ago protein is clearly visible and consistent with the expected size of 90.4kDa, and the purification by Ni column affinity chromatography shows that the target protein is better purified.
In FIG. 1, c is pET-23a-PfWestern blot verification results of Ago induced expression products based on anti-His primary antibodies, wherein M is protein marker,1 is uninduced group, and 2 is IPTG induced group. The figure further demonstrates His-PfSuccessful expression of Ag protein, notably, his-PfAgo protein expression due to pET23a-PfAgo presents background expression.
In addition, the induction expression condition optimization experiment shows that the induction time is optimal about 20 hours, and the concentration of IPTG has little influence on the protein expression quantity.
（2）His-PfIdentification of Ago fusion protein endonuclease Activity
Setting up a reaction system: 5' -phosphorylating the synthesized positive gDNA and adding His-PfAgo protein (1:350 molar ratio), 0.5pmol single-stranded target tDNA (target DNA, tDNA) andPfago reaction buffer (containing 200mM HEPES ph= 8.0,2.5M sodium chloride and 5mM manganese chloride) was reacted at 95 ℃ for 30min. Specifically, gDNA mediationPfAgo eggA schematic of the white cut tDNA is shown in FIG. 2a, where the arrow indicates the cleavage site.
After the reaction is finished, adding a 2 XTBE-PAGE electrophoresis loading buffer, incubating for 5min at 95 ℃, and performing 20% TBE-PAGE electrophoresis, and performing light-proof dyeing on SYBR Gold nucleic acid dye for 3-5 min to verify the digestion condition. The results are shown in FIG. 2 b, where 1 is that the system contains only tDNA and 2 is that noPfAgo protein control, 3 was the gDNA control without 5' phosphorylation and 4 was the result of the target sample cleavage. Indicating 5' phosphorylation mediated by gDNAPfCleavage of 45nt single-stranded tDNA by Ago protein resulted in 34nt DNA product, thus purified His-PfAgo fusion proteins have gDNA-mediated activity in cleaving DNA.
Example 1
The target sequence in this example is a PCR product amplified by primer pair B19-NS1-F/R (sequence shown as SEQ ID NO: 5-6), designated as B19 NS-p. The designed and synthesized gDNA of 3 targeted B19 NS-p are respectively marked as gt-B19 (the sequence is shown as SEQ ID NO: 1), gf-B19 (the sequence is shown as SEQ ID NO: 2) and gr-B19 (the sequence is shown as SEQ ID NO: 3). The sequence of the corresponding molecular beacon MB-B19 is shown as SEQ ID NO. 4.
The reaction detection system is constructed as follows:
5' -phosphorylating the three gDNA-B19 strips, respectively, and then adding His-PfAgo protein, 0.5pmol B19 NS-p,Pfago reaction buffer, 5pmol molecular beacon MB-B19. In the reaction system, three gDNA and His-PfThe molar ratio of the Ago proteins is 1:460.
The working principle of the reaction system is shown in figure 3: three gDNA-B19 guidesPfAgo cut B19 virusns1The gene double-stranded DNA is then used to cleave the molecular beacon MB-B19 using the produced gn-B19.
The reaction detection system was reacted at 95℃for 30min, and the results were shown in FIG. 4 by 20% TBE-PAGE and fluorescence detection. a is gDNA-B19 guidePfAgo specific cleavage of B19 Virusns1Gene product map (1, 3 are absentPfNegative control of Ago, 2, 4 are g-B19 (gr, gt, gf) +B19NS-p+PfAgo,1, 2 were SYBR Gold nucleic acid dye stained, 3, 4 were non-nucleic acid stained direct imaging), showing that B19 NS-p was in gIs cleaved under the mediation of DNA-B19 (gr, gt, gf) to obtain 5'P-gn-B19 (shown as SEQ ID NO: 7), which is matched withPfAgo binding targets the 28nt fluorescence labeled molecular beacon MB-B19 yielding 16nt DNA product. b is an analysis chart of the fluorescence intensity detection result, and showsPfThe fluorescence value after Ago cleavage of the molecular beacon MB-B19 was approximately zeroPf2.3 times that of the Ago control group indicated that cleavage of molecular beacons could be achieved in this reaction.
Therefore, if the electrophoresis detects 16nt products or the fluorescence value is increased relative to the negative control, the reaction detection system indicates that the B19 virus exists in the sample to be detected.
Sensitivity detection: according to the reaction detection system, the B19 virus added into the reaction system is reduced in a gradient mannerns1The amount of PCR product (B19 NS-p) of the gene, and the content of other substances in the system is unchanged; after reaction at 95℃for 30min, the reaction set was checked with and without addition ofPfFluorescence value for Ago control. The results are shown in FIG. 5: the increase in fluorescence value was still detectable at 0.1pmol of PCR product contained in the system.
Example 2
The detection system established in example 1 requires three guide mediations, increasing the complexity of the reaction. Considering the 5 'phosphorylation of g-MB-B19 and its pairing with MB-B19, which is generated by the first round of cleavage, this example allows for amplification of a novel tDNA (designated as B19 NS-p', the amplification primers of which are shown in SEQ ID NO:1 and SEQ ID NO: 6) by gf-B19 alonePfThe Ago cuts to obtain a first round product (marked as g-MB-B19 and shown as sequence SEQ ID NO: 8), thereby simplifying the reaction system and the working principle is shown as figure 6.
Set up targeting B19 virusns1Reaction System of Gene PCR product (B19 NS-p'): 5 '-phosphorylated gf-B19 (i.e., 5'P-gf-B19) andPfago fusion protein (molar ratio 1:460), 0.5pmol B19 NS-p',5pmol molecular beacon MB-B19 (same as in example 1)PfAfter being mixed, the Ago reaction buffer solution is placed at 95 ℃ for reaction for 30min, and is detected by 20% TBE-PAGE electrophoresis and fluorescence intensity detection.
The result of the electrophoresis detection is shown in FIG. 7 a (in the figure, 1 is nothingPfNegative control of Ago, 2 was gf-B19+B19NS-p' +PfAgo, left panel is SYBR Gold nucleic acid dye staining, right panel is uv gel imager direct imaging) shows: b19 NS-p ' is cleaved under the mediation of single 5'P-gf-B19 to obtain 5'P-g-MB-B19, which is further compared withPfAgo binding targets the 28nt fluorescence labeled molecular beacon MB-B19 yielding 16nt DNA product.
The fluorescence intensity detection results are shown in figure 7 b,Pfthe fluorescence value after Ago cleavage of the molecular beacon MB-B19 was approximately zeroPf2.4 times that of the Ago control group. Indicating single gDNA mediatedPfThe Ago detection system can also be used for the nucleic acid detection of B19 virus.
Sensitivity detection: according to the reaction detection system, the B19 virus added into the reaction system is reduced in a gradient mannerns1The amount of PCR product (B19 NS-p') of the gene, and the content of other substances in the system is unchanged; after reaction at 95℃for 30min, the reaction set was checked with and without addition ofPfFluorescence value for Ago control. The results show that the amount of target PCR product of the system can reach the level of 0.1pmol (see FIG. 8).
Comparative example 1
Unlike example 1, this example alters the gDNA sequence and its targeted B19 virusns1A gene fragment. In this example, three guide are designated as gt-B19-1 (SEQ ID NO: 11), gf-B19-1 (SEQ ID NO: 12) and gr-B19-1 (SEQ ID NO: 13), respectively, the first round of cleavage product is designated as gn-B19-1 (SEQ ID NO: 14) and the molecular signature is designated as MB-B19-1 (SEQ ID NO: 15).
Comparative example 2
Unlike example 1, this example alters the gDNA sequence and its targeted B19 virusns1A gene fragment. In this example, three guide are designated as gt-B19-2 (SEQ ID NO: 16), gf-B19-2 (SEQ ID NO: 17) and gr-B19-2 (SEQ ID NO: 18), respectively, the first round of cleavage product is designated as gn-B19-2 (SEQ ID NO: 19) and the molecular signature is designated as MB-B19-2 (SEQ ID NO: 20).
The detection results of comparative examples 1 and 2 are shown in FIG. 9, wherein lanes 2-7 are sequentially guided by gn-B19, gn-B19-1 and gn-B19-2 to cleave the corresponding molecular beacons, and lanes 9-10 are sequentially guided by three groups of guide to cleave the PCR products followed by a second round of cleavage of the molecular beacons. As can be seen from the figure, gn-B19 in example 1 can achieve cleavage of molecular beacon MB-B19, whereas cleavage in comparative examples 1, 2 is poor in effect, which is unfavorable for detection.
In conclusion, the invention adoptsPfAgo-mediated nucleic acid detection technology successfully realizes three or single gDNA guidancePfAgo targeting B19 virusns1, the nucleic acid detection of the conservation area has low detection cost and high sensitivity. The method provides an important tool for pathogen detection of other parvoviruses such as canine parvovirus (Canine parvovirus, CPV), porcine parvovirus (Porcine parvovirus, PPV) and the like, and also provides important support for monitoring and preventing infectious diseases of pathogenic microorganisms of human beings and animals and epidemic diseases, thereby having important practical application value.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
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Claims (6)
1. A test kit comprising:
the gDNA guiding PfAgo protein to target the ns1 gene of the B19 virus is specifically shown as a gt-B19 shown as a sequence SEQ ID NO. 1, a gf-B19 shown as a sequence SEQ ID NO. 2 and a gr-B19 shown as a sequence SEQ ID NO. 3, or only a gf-B19 shown as a sequence SEQ ID NO. 2;
a molecular beacon with a fluorescent group and a quenching group, wherein the sequence of the molecular beacon is shown as SEQ ID NO. 4;
pfago protein;
an amplification reagent for specifically amplifying a target sequence on the ns1 gene, wherein the amplification reagent comprises a primer pair with a sequence shown as SEQ ID NO. 5-6;
the detection kit is used for identifying or assisting in identifying the B19 virus, and the identification or assisting in identifying is used for non-disease diagnosis and treatment.
2. A PfAgo protein mediated B19 virus nucleic acid detection method not aimed at diagnosis and treatment of a disease, characterized in that the detection is performed using the detection kit of claim 1, and the detection comprises the steps of:
s1, amplifying a sample to be detected to obtain tDNA;
s2, setting up a reaction system comprising 5' -phosphorylated gDNA, pfAgo protein, tDNA, pfAgo reaction buffer and molecular beacons, and performing a cleavage reaction;
and S3, after the reaction is finished, performing fluorescence detection on the reaction system.
3. The method for detecting nucleic acid of PfAgo protein mediated B19 virus according to claim 2, wherein in the reaction system of step S2, the molar ratio of gDNA to PfAgo protein is 1:460.
4. The method for detecting the nucleic acid of the Pfago protein mediated B19 virus according to claim 2, wherein the temperature of the cleavage reaction is 90-98 ℃.
5. The method for detecting a PfAgo protein mediated B19 viral nucleic acid according to claim 4, wherein the cleavage reaction is performed at a temperature of 95 ℃.
6. The method for detecting the nucleic acid of the Pfago protein-mediated B19 virus according to claim 2, wherein the tDNA content in the reaction system in the step S2 is not less than 0.1pmol.
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