CN112126714A - Coronavirus detection product and application thereof - Google Patents
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Abstract
The invention relates to the technical field of molecular biology, in particular to a coronavirus detection product and application thereof, wherein a detection substance comprises a primer and a probe for specifically identifying coronavirus nucleic acid, the primer comprises an upstream primer and a downstream primer, and the upstream primer is selected from the group consisting of SEQ ID NO:1 or the first upstream primer as shown in SEQ ID NO: 2; the downstream primer is selected from the group consisting of SEQ ID NO: 3 or the first downstream primer as set forth in SEQ ID NO: 4, a second downstream primer; the probe is selected from the group consisting of SEQ ID NO: 5 or the first probe as set forth in SEQ ID NO: 6, and a second probe. The coronavirus detection product provided by the invention solves the current situation of the detection reagent for needle-free oropharyngeal swabs, nasopharynx swabs, saliva, sputum, excrement and blood samples for the first time, qualitatively detects the coronavirus total viruses, and prevents other coronaviruses except new coronaviruses from being missed.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a coronavirus detection product and application thereof.
Background
Coronaviruses belong to the phylogenetic group of Coronaviridae (Coronaviridae) coronaviruses (Coronavirus). The coronavirus is a positive strand single strand RNA virus with an outer mantle (envelope), the genetic material of the coronavirus is the largest of all RNA viruses, the pathogenicity of different types of viruses is different, the clinical manifestations are different, and the symptom caused by the OC43 strain is generally more serious than that caused by 229E virus. Coronavirus is one of the main pathogens of the common cold in adults, and the latent period of infection is generally 2-5 days. Typical coronavirus infection is a common cold symptom such as runny nose and discomfort, and symptoms such as fever, shivering and vomiting can also appear. Representative variants are mainly the causative agent of atypical pneumonia (SARS) and the novel coronavirus (nCoV-2019).
Detection of coronaviruses currently there are two most commonly used methods in the clinic: firstly, a suspected patient is firstly detected whether to be infected by other influenza viruses, if not, other projects are continuously screened, such as novel coronavirus (or influenza virus) detection, the method is time-consuming and labor-consuming, the hospitalizing time of the patient is increased, and the risk of missed detection of other coronavirus is easily caused, such as the patient is infected by other coronavirus, or the selected detection gene locus is mutated by the novel coronavirus; secondly, for suspected patients, a plurality of items are detected at one time to screen pathogens, but the detection cost for the patients is too high, and the problem of missed detection is still not solved.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a coronavirus detection product and its use, which solve the problems of the prior art.
In order to achieve the above objects and other related objects, the present invention provides in a first aspect a fluorescent PCR detection material for coronavirus, the detection material comprising a primer and a probe that specifically recognizes coronavirus nucleic acid, the primer comprising an upstream primer and a downstream primer, the upstream primer being selected from the group consisting of: as shown in SEQ ID NO:1 or the first upstream primer as shown in SEQ ID NO: 2; the downstream primer is selected from: as shown in SEQ ID NO: 3 or the first downstream primer as set forth in SEQ ID NO: 4, a second downstream primer; the probe is selected from the group consisting of SEQ ID NO: 5 or the first probe as set forth in SEQ ID NO: 6, and a second probe.
The second aspect of the invention provides the application of the coronavirus fluorescent PCR detection substance in preparing a coronavirus detection product.
The third aspect of the invention provides a coronavirus fluorescent PCR detection kit, which comprises a PCR detection mixture, wherein the PCR detection mixture comprises the coronavirus fluorescent PCR detection substance.
As described above, the coronavirus detection product and the application thereof have the following beneficial effects:
1) clinical specimen type comprehensive coverage: including nasopharyngeal swabs, oropharyngeal swabs, saliva, sputum, stool, and blood samples; the current situation that no Coronavir detection reagent for nasopharyngeal swabs, oropharyngeal swabs, saliva, sputum, excrement and blood samples is available on the market is solved for the first time.
2) Targeted detection of coronavirus total viruses: preventing missing detection caused by other types of coronavirus except new corona or missing detection caused by mutation;
3) the single test mode of operation greatly reduces the patient's hospitalization time and the exposure risk during the hospitalization waiting process.
4) The fluorescent quantitative PCR technology can realize real-time detection, qualitative analysis and high detection flux, and has the advantages of simple and convenient operation, high sensitivity and good specificity.
Drawings
FIG. 1 is a schematic diagram of a pre-loaded plate of the RNA nucleic acid extraction reagent of the present invention.
FIG. 2 shows a PCR amplification chart comparing the RNA nucleic acid extraction effect of the saline-diluted sample according to the present invention.
FIG. 3 shows a PCR amplification chart comparing the RNA nucleic acid extraction effect of the diluted sample of the negative nasopharyngeal swab washing solution of the present invention.
FIG. 4 shows the results of sensitivity analysis of the fluorescent PCR detection substance for coronavirus of the present invention.
FIG. 5 shows the result of the specificity analysis of the fluorescent PCR detection substance for coronavirus of the present invention.
Detailed Description
The invention provides a coronavirus fluorescent PCR detection substance, which comprises a primer and a probe for specifically recognizing coronavirus nucleic acid, wherein the primer comprises an upstream primer and a downstream primer, and the upstream primer is selected from the following primers: as shown in SEQ ID NO:1 or the first upstream primer as shown in SEQ ID NO: 2; the downstream primer is selected from: as shown in SEQ ID NO: 3 or the first downstream primer as set forth in SEQ ID NO: 4, a second downstream primer; the probe is selected from the group consisting of SEQ ID NO: 5 or the first probe as set forth in SEQ ID NO: 6, and a second probe.
Preferably, the upstream primer comprises: as shown in SEQ ID NO:1 and a first upstream primer shown as SEQ ID NO: 2; the downstream primer comprises the sequence shown in SEQ ID NO: 3 and the first downstream primer as set forth in SEQ ID NO: 4, a second downstream primer; the probe comprises the nucleotide sequence shown as SEQ ID NO: 5 and a first probe as set forth in SEQ ID NO: 6, and a second probe. The two pairs of primers can avoid the condition of missing detection, but only one pair of primers can detect the existence of the coronavirus.
Specifically, the Coronavirus belongs to the genus Coronavir (Coronavir), the diameter of the Coronavirus is about 80-120 nm, the virus of the genus Coronavir is a plus-strand single-stranded RNA virus with an outer mantle (envelope), the 5 'end of a genome has a methylated cap structure, the 3' end of the genome has a poly (A) tail, the total length of the genome is about 27-32kb, and the Coronavirus is a virus with the largest genome in the conventional RNA viruses. The coronavirus includes common coronavirus 229E, OC43 and SARS coronavirus (SARS-CoV)3 types.
In particular, the method comprises the following steps of,
a first upstream primer: 5'-GTGTCTTTCTGCTATATGC-3' (SEQ ID NO: 1);
a second upstream primer: 5'-TGCCGTCTTAGATATGTG-3' (SEQ ID NO: 2);
first downstream primer: 5'-GTCTGTCAACAAATGGAC-3' (SEQ ID NO: 3);
a second downstream primer: 5'-TGGTGTAAACTCATCTTCTA-3' (SEQ ID NO: 4);
a first probe: 5'-CCTTCTAAGTCAGTACCAGCGTGT-3' (SEQ ID NO: 5);
a second probe: 5'-TGCTACCAAGGATAGTACGACCATTC-3' (SEQ ID NO: 6).
One end of the first probe or the second probe is marked with a fluorescence reporter group, and the other end is marked with a marked fluorescence quenching group.
Preferably, the first probe or the second probe is labeled with a fluorescent reporter group at the 5 'end and a labeled fluorescent quencher group at the 3' end.
Specifically, the fluorescent reporter group is selected from one of the following: FAM, HEX, VIC, CY3, ROX, 610, TEXAS RED, CY 5.
Specifically, the fluorescence quenching group corresponds to a fluorescence reporter group, and can quench the corresponding reporter group. For example, when the fluorescence reporter is FAM, the fluorescence quencher is selected from one of the following: BHQ1, Dabcyl, QYS-7, BHQ 2.
Preferably, the fluorescence reporter group is a FAM fluorophore group, and the fluorescence quencher group is BHQ 1.
The second aspect of the invention provides the application of the coronavirus fluorescent PCR detection substance in preparing a coronavirus detection product.
The coronavirus detection product takes one or more of nasopharyngeal swab, oropharyngeal swab, saliva, sputum, feces or blood as a detection sample source. The clinical sample type of the coronavirus detection product is comprehensively covered, and is not limited by the sample type.
The coronavirus detection product is used for judgment of coronavirus, selection of a treatment scheme, and/or prognosis evaluation.
In a third aspect, the invention provides a fluorescent PCR detection kit for coronavirus, which comprises a PCR detection mixture, wherein the PCR detection mixture contains the aforementioned primer and probe.
In one embodiment, the concentration of the primers and probes in the PCR assay mixture are both 20. mu. mol/L.
Preferably, the kit further comprises an internal standard, and a reagent and an internal standard probe for specifically recognizing the internal standard. The reagent for specifically identifying the internal standard comprises primers for specifically amplifying the internal standard, and the primers for specifically amplifying the internal standard comprise an internal standard upstream primer and an internal standard downstream primer.
Further, the internal standard probe, the internal standard upstream primer and the internal standard downstream primer are mixed in the PCR detection mixture. In one embodiment, the concentrations of the internal standard probe, the internal standard upstream primer and the internal standard downstream primer in the PCR detection mixture are all: 20. mu. mol/L.
In a preferred embodiment, the nucleotide sequence of the internal standard upstream primer is 5'-CCCGGTTTCTATAAATTGAGC-3' (SEQ ID NO: 7).
In a preferred embodiment, the nucleotide sequence of the internal standard downstream primer is 5'-GCGACGCAAAAGAAGATG-3' (SEQ ID NO: 8).
In a preferred embodiment, the nucleotide sequence of the internal standard probe is as shown in SEQ ID NO: 9, in particular, 5'-CGCTCTCTGCTCCTCCTGTT-3' (SEQ ID NO: 9). One end of the internal standard probe is marked with a fluorescence reporting group, and the other end of the internal standard probe is marked with a marked fluorescence quenching group. Preferably, the first probe or the second probe is labeled with a fluorescent reporter group at the 5 'end and a labeled fluorescent quencher group at the 3' end. Preferably, the fluorescent reporter group marked by the internal standard probe is a HEX fluorescent group; the fluorescence quenching group is BHQ 1.
Preferably, the detection mixture further comprises PCR MIX and H2O。
In one embodiment, the PCR MIX is a commercially available product. For example, it may be a product manufactured by Life Technologies, under the name AGPATH-ID ONE-STEP RT-PCR, cat # 4387291. In one embodiment, the PCR MIX and H2The usage amount of O is as follows: PCR MIX 15. mu.l/test; h2O 6μL/test。
In one embodiment, the total volume of the PCR assay mix tested is 24.41 μ L, and the volumes of each substance in the PCR assay mix are as follows:
the components of the PCR detection mixture can be enlarged in proportion, and the experimental effect is not influenced.
Preferably, the kit further comprises one or more of the following components: PCR enzyme, negative quality control product, positive quality control product and internal standard.
In one embodiment, the PCR enzymes include Taq enzyme and reverse transcriptase.
In one embodiment, the negative quality control substance is selected from the group consisting of RNase-free H2And O. The RNase-free H2O can be DEPC-H2O。
Preferably, the internal standard is prepared from an internal standard plasmid.
Preferably, the positive control is prepared from Coronavir plasmid or pseudovirus.
In one embodiment, the Coronavir plasmid or pseudovirus is a plasmid or pseudovirus comprising a target fragment of SEQ ID NO.10 or SEQ ID NO. 11. The internal standard plasmid is a plasmid containing a target fragment shown in SEQ ID NO. 12 sequence.
In one embodiment, when a 40 μ L reaction system is prepared in the kit, 20 μ L of the PCR assay mixture is mixed with 0.5 μ L of the PCR enzyme, and after mixing, 20 μ L of the PCR assay mixture is mixed with 20 μ L of the sample to be tested, so as to obtain 40 μ L of the PCR reaction system.
Preferably, the 0.5 μ L PCR enzyme is prepared by mixing 0.34 μ L Taq enzyme 5U/μ L and 0.16 μ L reverse transcriptase.
Specifically, the to-be-detected sample is an RNA template solution, a negative quality control product and a positive quality control product of the sample.
PCR reaction threshold setting for the present application: the threshold value setting principle is that the threshold value line just exceeds the highest point of the fluorescence curve of the negative quality control product detection.
The quality control of the PCR detection result of the invention is as follows: the detection result of the negative quality control product is as follows: the FAM channel Ct column shows either underwritered (ABI7500) or NoCt (MIC QPCR), VIC/HEX channel Ct values less than 36. The criteria for judging the test results are as follows:
TABLE 1 interpretation of the test results
Preferably, the kit further comprises a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent is a magnetic bead method nucleic acid extraction reagent provided by the application.
The magnetic bead method nucleic acid extraction reagent comprises lysis solution, wherein the lysis solution at least comprises the following components in percentage by weight based on the total amount of the lysis solution:
specifically, based on the total amount of the lysis solution, in the lysis solution,
the content of guanidinium isothiocyanate may be 1-3mol/L, 1-1.5mol/L, 1.5-2mol/L, 2-2.5mol/L, 2.5-3 mol/L.
The sodium lauryl sulfate may be present in an amount of 0.05-0.1% (w/v), 0.05-0.06% (w/v), 0.06-0.07% (w/v), 0.07-0.08% (w/v), 0.08-0.09% (w/v), 0.09-0.1% (w/v).
The NP40 content may be 1-5% (w/v), 1-2% (w/v), 2-3% (w/v), 3-4% (w/v), 4-5% (w/v).
The content of Tris-HCl can be 10-80mmol/L, 10-20mmol/L, 20-30mmol/L, 30-40mmol/L, 40-50mmol/L, 50-60mmol/L, 60-70mmol/L, 70-80 mmol/L.
The content of EDTA can be 5-15mmol/L, 5-8mmol/L, 8-10mmol/L, 10-12mmol/L, 12-15 mmol/L.
The volume percentage of the beta-mercaptoethanol can be 0.1-0.5%, 0.1-0.2%, 0.2-0.3%, 0.3-0.4%, 0.4-0.5%.
Wherein NP40 is ethyl phenyl polyethylene glycol.
The solvent may be process water.
In one embodiment, the lysis solution comprises at least the following components, wherein the content of each component is based on the total amount of the lysis solution:
the lysate can be used for extracting coronavirus nucleic acid from nasopharyngeal swabs, oropharyngeal swabs, saliva, sputum, feces and blood samples.
The pH value of the lysate is 8.5 +/-0.1.
The substance for extracting coronavirus nucleic acid by the magnetic bead method further comprises one or more of magnetic beads, washing liquid, eluent, RNA (ribonucleic acid) precipitation-assisting agent and proteinase K.
The RNA assistant and proteinase K are commercially available products, and can be, for example: RNA precipitation aid (Glycogen,20mgml), proteinase K, Tiangen Biochemical technology (Beijing) Ltd., cat # RT 403.
In one embodiment, the magnetic beads are used at a concentration of 50-100 mg/mL.
The magnetic bead can be in the model of Orrunn SM 1-050.
The first washing solution is used for washing away protein impurities in the sample. In one embodiment, the washing solution comprises a first washing solution, wherein the first washing solution comprises at least the following components, wherein the components are contained in the following amounts based on the total amount of the first washing solution: guanidine isothiocyanate: 0.5-2mol/L, sodium chloride: 0.1-0.5mol/L, triton X-100: 0.5-1% (w/v), the volume percentage of absolute ethyl alcohol is 30-50%, Tris-HCl: 2-8mmol/L, and the solvent is water.
The second washing liquid is used for washing salt impurities. In one embodiment, the washing solution further comprises a second washing solution, wherein the second washing solution contains at least the following components in the following amounts based on the total amount of the second washing solution: anhydrous ethanol: the volume percentage is 75 percent, and the solvent is water.
The eluent is used for eluting nucleic acid. The eluent may also be a commercially available product. In one embodiment, the eluent comprises at least the following components, wherein the content of each component is based on the total amount of the eluent: 10-80mmol/L Tris-HCL, 0.5-25mmol/L EDTA and water as solvent.
The pH of the first wash solution was 8.0. + -. 0.1.
The pH of the eluent is 8.0 plus or minus 0.1.
When in use, the volume ratio of the lysis solution to the magnetic beads is (300-: 10 mu L of the solution; .
The lysis solution and the magnetic beads can be mixed and then are pre-loaded in a 96-well plate. The 96-well plate is selected from a 96-deep-well plate.
In one embodiment, as shown in FIG. 1, the lysate is pre-loaded with magnetic beads in row A of a 96-well plate and the volume is controlled to 500. mu.L. + -. 50. mu.L.
The first washing solution, the second washing solution and the eluent can be respectively pre-filled in a 96-well plate.
In one embodiment, the first wash solution is pre-loaded in a 96-well plate in row B, C and the volume is controlled to 600. mu.L. + -. 50. mu.L.
In one embodiment, the second wash solution is pre-loaded in a 96-well plate in row D, E, and the volume is controlled to 500. mu.L. + -. 50. mu.L.
In one embodiment, the eluate is prepackaged in a 96-well plate in H rows and the volume is controlled at 50 μ L. + -. 5 μ L.
The kit also comprises a virus inactivator, wherein the virus inactivator can be a commercial chlorine-containing disinfectant, peracetic acid, chloroform or other lipid solvents.
The virus inactivating agent can inactivate coronavirus.
The coronavirus is collected from a patient infected or suspected to be infected with the coronavirus.
The sample type is selected from one or more of nasopharyngeal swab, oropharyngeal swab, saliva, sputum, feces and blood.
If the kit is matched with a coronavirus standard substance, quantitative detection can be realized.
In a fourth aspect, the invention provides a method of using the kit, the method comprising the steps of:
1) inactivating the sample;
2) extracting RNA of a sample to be detected to obtain an RNA template solution;
3) adding sample and preparing a reaction system;
4) and (4) PCR detection.
The kit of the invention aims at qualitative detection of coronavirus nucleic acid, adopts the Taqman fluorescence quantitative PCR principle, designs specific primers aiming at coronavirus genes (nucleotide sequences are shown as SEQ ID NO:10 and SEQ ID NO: 11), amplifies specific nucleic acid sequences, simultaneously designs Taqman probes, and marks different fluorescence reporter groups which are positioned between an upstream primer and a downstream primer.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Example 1 design and Synthesis of primer probes for nasopharyngeal, oropharyngeal swabs, saliva, sputum, stool, and blood samples Coronavir detection kits
FAM fluorophores are marked on the 5 'of the Coronavir probe, and HEX fluorophores are marked on the 5' of the internal standard probe. Primers and probes for PCR detection of Coronavir (nucleotide sequences shown as SEQ ID NO:10 and SEQ ID NO: 11) and an internal standard (nucleotide sequence shown as SEQ ID NO: 12) are synthesized by the prior art.
Coronavirus：
A first upstream primer: 5'-GTGTCTTTCTGCTATATGC-3' (SEQ ID NO: 1);
a second upstream primer: 5'-TGCCGTCTTAGATATGTG-3' (SEQ ID NO: 2)
First downstream primer: 5'-GTCTGTCAACAAATGGAC-3' (SEQ ID NO: 3);
a second downstream primer: 5'-TGGTGTAAACTCATCTTCTA-3' (SEQ ID NO: 4)
A first probe: 5 '-FAM-CCTTCTAAGTCAGTACCAGCGTGT-BHQ 1-3' (SEQ ID NO: 5);
a second probe: 5 '-FAM-TGCTACCAAGGATAGTACGACCATTC-BHQ 1-3' (SEQ ID NO: 6)
Internal standard:
upstream primer 5'-CCCGGTTTCTATAAATTGAGC-3' (SEQ ID NO: 7)
Downstream primer 5'-GCGACGCAAAAGAAGATG-3' (SEQ ID NO: 8)
Probe 5 '-HEX-CGCTCTCTGCTCCTCCTGTT-BHQ 1-3' (SEQ ID NO: 9)
Example 2 comparison of extraction efficiency of the present invention with other commercial extraction kits
1. Experimental methods
1.1 detection of objects
Constructing a high-concentration Coronavir positive simulation sample, wherein the positive simulation sample consists of a synthetic product of a new Coronavirus genome sequence (shown as SEQ ID NO.10 and SEQ ID NO. 11) and a background matrix of normal human exfoliated cells, and taking the high-concentration Coronavir positive simulation sample, and diluting the high-concentration Coronavir positive simulation sample by three gradients of a Coronavir negative nasopharyngeal swab washing liquid sample and normal saline respectively. Three concentration samples diluted by Coronavir negative nasopharyngeal swab wash samples are respectively defined as XL-1, XL-2 and XL-3, and three concentration samples diluted by physiological saline are respectively defined as XN1, XN2 and XN 3.
1.2 inactivation of samples
And (3) adding 1.5mL or an equal volume of a virus inactivating agent into the samples, wherein the virus inactivating agent comprises Chelex: 0.1% (w/v), NP 40: 5% (w/v), Tris-HCL: 40mmol/L, pH8.5 + -0.1, shaking, mixing, and standing at room temperature for 30 min.
1.3 sample nucleic acid extraction
Sample extractions were performed using the self-contained nucleic acid RNA extraction reagents of the invention and other company RNA extraction reagents on the market (Qiagen, Cat. 52904), with two replicates per extraction. Other companies extracted the reagents according to their instructions. The components of the extraction reagent are pre-arranged in a 96-deep-well plate, the components and the volume of each column of the deep-well plate are shown in the attached figure 1, and two multiple wells are extracted from each sample. The extraction reagent comprises the following components:
based on the total amount of the cracking solution, the content of each component of the cracking solution is as follows:
based on the total amount of the first washing liquid, the content of each component of the first washing liquid is as follows: guanidine isothiocyanate: 2mol/L, sodium chloride: 0.5mol/L, triton X-100: 1% (w/v), absolute ethanol: 50% (v/v), Tris-HCl: 2mmol/L, and the solvent is water.
And the content of each component of the second washing liquid is as follows by taking the total amount of the second washing liquid as a reference: anhydrous ethanol: 75% (v/v) and the solvent is water.
Based on the total amount of the eluent, the contents of all the components of the eluent are as follows: 40mmol/L of EDTA, 10mmol/L of EDTA and water as solvent.
Adding 1 mu L of internal standard in the line A of the prepackage plate, and then sequentially adding XL-1, XL-2, XL-3, XN1, XN2, XN3, a negative quality control product and a positive quality control product into each hole. The deep well plate was placed in a fully automatic nucleic acid extractor (model: EX3600) of Biomedicine technology, Inc., of Shanghai, and the program "RNA Isolation" was run, and after the program was run, the RNA template solution in line H was taken out by a pipette gun for use.
1.4 sample detection
When preparing PCR reaction liquid, taking 20 mu L of Coronavir PCR detection mixture, mixing with 0.5 mu L of PCR enzyme, shaking and uniformly mixing for several seconds, centrifuging at 3000rpm for several seconds, taking 20 mu L, placing in a PCR tube, then adding 20 mu L of sample, negative quality control product or nucleic acid extraction product solution of the positive quality control product into the PCR reaction tube, covering a PCR reaction tube cover, and immediately carrying out PCR amplification reaction.
The cycle parameters are set as: 10min at 50 ℃; 2min at 95 ℃; 45 cycles of 95 ℃ X15 sec and 60 ℃ X30 sec were alternately repeated. Single-point fluorescence detection was performed at 60 ℃ in a 40. mu.L reaction system.
The fluorescence channel was selected as in table 2 below.
TABLE 2 selection of fluorescent channels
| Model type | Channel | Channel | |
| ABI7500 | FAM | VIC | |
| | Channel | 1 | |
Note: if an ABI Prism7500 real-time fluorescent PCR instrument is used, both the passive reference and the query will need to be selected as "none".
2. Results of the experiment
The amplification curve of the nasopharyngeal swab washing liquid sample diluted by normal saline is shown in the attached figure 2, the amplification curve of the nasopharyngeal swab sample diluted by Coronavir positive nasopharyngeal swab washing liquid is shown in the attached figures 2 and 3, and the Ct value statistics of the detection results are shown in the table 3:
TABLE 3 comparative test results of extracted reagents
In FIG. 2, light color represents the extraction reagent of the present invention, dark color represents the extraction reagent of other companies, and the Ct value data of Table 2 shows that the two companies have no significant difference in the extraction efficiency of the sample diluted with physiological saline.
In the attached figure 3, the Ct value data in Table 3 show that the efficiency of extracting the sample diluted by the nasopharyngeal swab washing liquid from the reagent provided by the invention is not obviously different, but the peak of the curve is good for the product of this company.
The above results show that the extraction efficiency of the two extraction reagents is not obviously different for the sample diluted by normal saline, and the extraction effect of the extraction reagent of the invention is better than that of other commercial extraction reagents for the sample diluted by nasopharyngeal swab washing liquor. The fact that PCR inhibitors exist in nasopharyngeal swab washing liquid samples is proved, but the detection reagent has strong anti-interference capability and better performance of the extracted reagent, and the obtained RNA template samples obtain better PCR amplification effect.
Example 3 application of Coronavir combined detection kit in saliva and sputum
1. Experimental methods
1.1 detection of objects
Saliva and sputum obtained from 4 clinical samples were subjected to detection of Coronavir nucleic acid, and the 4 samples were identified as 2 Coronavir positive samples and 2 Coronavir negative samples by a hospital culture method, and numbered as S1, S2, S3 and S4.
1.2 sample inactivation and nucleic acid extraction
And (3) adding 1.5mL of virus inactivator into the samples respectively, shaking and uniformly mixing, and standing at room temperature for 30 min.
The extraction of 4 saliva and sputum samples was carried out using the extraction reagent of the present invention (same as 1.3 in example 2). Adding 1 mu L of internal standard in the row A of the prepackage plate, and then sequentially adding S1, S2, S3 and S4, and a negative quality control substance or a positive quality control substance in each well. The 96-well plate is placed in a full-automatic nucleic acid extractor, the program "RNA Isolation" is run, and after the program is run, the RNA template solution in row H is taken out by a pipette gun for standby.
1.3 sample detection
When preparing PCR reaction liquid, taking 20 mu L of Coronavir PCR detection mixture, mixing with 0.5 mu L of PCR enzyme to obtain PCR reaction mixed liquid, shaking and uniformly mixing for several seconds, centrifuging at 3000rpm for several seconds, taking 30 mu L of the mixture, placing the mixture into a PCR tube, adding 20 mu L of sample, negative quality control product or RNA template solution of positive quality control product into the PCR tube, covering the PCR tube, and immediately carrying out PCR amplification reaction.
The cycle parameters are set as: 10min at 50 ℃; 2min at 95 ℃; 45 cycles of 95 ℃ X15 sec and 60 ℃ X30 sec were alternately repeated. Single-point fluorescence detection was performed at 60 ℃ in a 40. mu.L reaction system.
The fluorescence channel was selected as shown in Table 2.
2. Results of the experiment
As can be seen from the data in table 4, the detection results of samples S1 and S3 were both Coronavirus positive, and samples S2 and S4 were Coronavirus negative. The detection result is consistent with the detection result of a culture method provided clinically, and the kit has better specificity.
TABLE 4 qualitative test results of the kit samples
Example 4 evaluation of the Performance of the Coronavir detection kit in sputum
1. Experimental methods
1.1 detection of objects
(1) Sensitivity analysis
Taking 1 part of Coronavir analog positive sample, diluting the sample into 5 gradients with sample diluent which is 10 times, sequentially numbering S1, S2, S3, S4 and S5, adding negative quality control substances and positive quality control substances, extracting nucleic acid and detecting.
The detection method comprises the following steps: and (3) mixing 30 mu L of PCR detection mixture with 0.5 mu L of PCR enzyme, shaking and uniformly mixing for several seconds, centrifuging at 3000rpm for several seconds, placing 30 mu L of the mixed liquid into a PCR tube, adding 20 mu L of a sample, a negative quality control product or a nucleic acid extraction product of the positive quality control product into the PCR reaction tube, covering a PCR reaction tube cover, and immediately carrying out PCR amplification reaction.
The cycle parameters are set as: 10min at 50 ℃; 2min at 95 ℃; 45 cycles of 95 ℃ X15 sec and 60 ℃ X60 sec were alternately repeated. Single-point fluorescence detection is carried out at 60 ℃ in a reaction system of 50 mu L.
(2) Specificity analysis
According to the specification of '2019-nCov nucleic acid detection reagent national reference substance', a non-target detection item for specific analysis is set for influenza virus, parainfluenza virus, Klebsiella pneumoniae, adenovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus enterprise reference substances and negative simulation swab samples. And (4) detecting after nucleic acid extraction.
The detection method comprises the following steps: and (2) mixing 30 mu L of PCR detection mixed solution with 0.5 mu L of PCR enzyme, uniformly mixing the mixture for several seconds by oscillation, centrifuging the mixture at 3000rpm for several seconds, placing 30 mu L of the mixed solution into a PCR tube, adding 20 mu L of nucleic acid extraction products of influenza virus, parainfluenza virus, Klebsiella pneumoniae, adenovirus, mycoplasma pneumoniae, chlamydia pneumoniae, respiratory syncytial virus enterprise reference products and negative simulation swab samples into the PCR reaction tube, covering a PCR reaction tube cover, and immediately performing PCR amplification reaction.
The cycle parameters are set as: 10min at 50 ℃; 2min at 95 ℃; 45 cycles of 95 ℃ X15 sec and 60 ℃ X60 sec were alternately repeated. Single-point fluorescence detection is carried out at 60 ℃ in a reaction system of 50 mu L.
2. Results of the experiment
2.1 sensitivity analysis: as shown in FIG. 4, the linear range of the results of S1, S2, S3, S4 and S5 was good, and the lowest detection limit of Coronavir was 450copies/mL when S5 was diluted by 4 gradients.
2.2 specific analysis: as shown in fig. 5, the results of the influenza virus, parainfluenza virus, klebsiella pneumoniae, adenovirus, mycoplasma pneumoniae, chlamydia pneumoniae, respiratory syncytial virus enterprise reference and negative mock swab samples were all negative, and the positive ginseng appeared normally, indicating that the reagent specificity was good.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the method of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
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Claims (10)
1. A fluorescent PCR detection material for coronavirus, wherein the detection material comprises a primer and a probe that specifically recognizes coronavirus nucleic acid, the primer comprises an upstream primer and a downstream primer, and the upstream primer is selected from the group consisting of: as shown in SEQ ID NO:1 or the first upstream primer as shown in SEQ ID NO: 2; the downstream primer is selected from: as shown in SEQ ID NO: 3 or the first downstream primer as set forth in SEQ ID NO: 4, a second downstream primer; the probe is selected from the group consisting of SEQ ID NO: 5 or the first probe as set forth in SEQ ID NO: 6, and a second probe.
2. The fluorescent PCR detection material for coronavirus according to claim 1, wherein the upstream primer comprises: as shown in SEQ ID NO:1 and a first upstream primer shown as SEQ ID NO: 2; the downstream primer comprises the sequence shown in SEQ ID NO: 3 and the first downstream primer as set forth in SEQ ID NO: 4, a second downstream primer; the probe comprises the nucleotide sequence shown as SEQ ID NO: 5 and a first probe as set forth in SEQ ID NO: 6, and a second probe.
3. The fluorescent PCR detection substance for coronavirus according to claim 1, wherein the first probe or the second probe is labeled with a fluorescent reporter group at one end and a fluorescent quencher group at the other end.
4. Use of a fluorescent PCR assay substance for coronavirus according to any one of claims 1-3 in the preparation of a coronavirus assay product.
5. The use according to claim 4, further comprising one or more of the following features:
1) the coronavirus detection product takes one or more samples of nasopharyngeal swabs, oropharyngeal swabs, saliva, sputum, feces or blood as a detection sample source;
2) the coronavirus detection product is used for judgment of coronavirus, selection of a treatment scheme, and/or prognosis evaluation.
6. A fluorescent PCR assay kit for coronavirus, wherein the kit comprises a PCR assay mixture, and the PCR assay mixture comprises the fluorescent PCR assay material for coronavirus according to any one of claims 1-3.
7. The coronavirus fluorescent PCR detection kit of claim 6, further comprising one or more of the following characteristics:
1) in the PCR detection mixture, the concentration of each primer and each probe is 20 mu mol/L;
2) the coronavirus fluorescent PCR detection kit also comprises an internal standard, a reagent for specifically identifying the internal standard and an internal standard probe;
3) the coronavirus fluorescent PCR detection kit also comprises one or more of the following components: PCR enzyme, negative quality control product, positive quality control product and internal standard.
8. The coronavirus fluorescent PCR detection kit of claim 7, further comprising one or more of the following characteristics:
a) the reagent for specifically identifying the internal standard in the characteristic 2) comprises primers of a specific amplification internal standard, and the primers of the specific amplification internal standard comprise an internal standard upstream primer and an internal standard downstream primer; the internal standard probe, the internal standard upstream primer and the internal standard downstream primer are mixed in a PCR detection mixture, and the nucleotide sequence of the internal standard upstream primer is shown as SEQ ID NO: 7, the nucleotide sequence of the internal standard downstream primer is shown as SEQ ID NO: 8, the nucleotide sequence of the internal standard probe is shown as SEQ ID NO: 9 is shown in the figure;
b) in the feature 3), the internal standard is prepared from an internal standard plasmid;
c) the negative quality control product is selected from RNase-free H2O；
d) The positive quality control product is prepared from coronavirus plasmid or pseudovirus.
9. The fluorescent PCR assay kit for coronavirus of claim 7, wherein the fluorescent PCR assay kit for coronavirus further comprises a nucleic acid extraction reagent, and the nucleic acid extraction reagent is a magnetic bead method nucleic acid extraction reagent.
10. The coronavirus fluorescent PCR detection kit of claim 9, wherein: the magnetic bead method nucleic acid extraction reagent comprises one or more of lysis solution, magnetic beads, washing solution, eluent, RNA (ribonucleic acid) precipitation-assisting agent and proteinase K.
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| CN111088405A (en) * | 2020-02-06 | 2020-05-01 | 苏州行知康众生物科技有限公司 | Primer probe composition, kit and method for detecting coronavirus 2019-nCoV |
| CN111118228A (en) * | 2020-03-31 | 2020-05-08 | 上海邦先医疗科技有限公司 | COVID-19 nucleic acid detection kit for novel coronavirus and use method thereof |
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| CN1829736A (en) * | 2003-04-10 | 2006-09-06 | 希龙公司 | The severe acute respiratory syndrome coronavirus |
| US20070270360A1 (en) * | 2003-04-15 | 2007-11-22 | Sirna Therapeutics, Inc. | Rna Interference Mediated Inhibition of Severe Acute Respiratory Syndrome (Sars) Gene Expression Using Short Interfering Nucleic Acid |
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| CN111088405A (en) * | 2020-02-06 | 2020-05-01 | 苏州行知康众生物科技有限公司 | Primer probe composition, kit and method for detecting coronavirus 2019-nCoV |
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