CN113549710A - Kit for rapidly and specifically detecting 2019 novel coronavirus and use method thereof - Google Patents
Kit for rapidly and specifically detecting 2019 novel coronavirus and use method thereof Download PDFInfo
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Abstract
The invention relates to a kit for rapidly and specifically detecting 2019 novel coronavirus (2019-nCoV) nucleic acid and a using method thereof, belonging to the technical field of biology. The kit comprises a working standard substance, a constant-temperature amplification reaction solution and a negative control substance, wherein the working standard substance comprises a positive control plasmid of a 2019 novel coronavirus (2019-nCoV) specific conserved sequence, and the specific conserved sequence is SEQ ID No. 1; the isothermal amplification reaction solution comprises 6 primers: an outer primer F3, the sequence of which is SEQ ID No. 2; an outer primer B3, the sequence of which is SEQ ID No. 3; the sequence of the inner primer FIP is SEQ ID No. 4; the sequence of the inner primer BIP is SEQ ID No. 5; the sequence of the loop primer LB is SEQ ID No. 6; and the sequence of the loop primer LF is SEQ ID No. 7. The negative control is nucleic acid extract of nasopharyngeal swab of healthy person. The 2019 novel coronavirus (2019-nCoV) nucleic acid isothermal amplification detection kit provided by the invention has the advantages of good consistency, high accuracy, quick operation and simple method.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a kit for quickly and specifically detecting 2019 novel coronavirus (2019-nCoV) nucleic acid and a using method thereof.
Background
2019A novel coronavirus (2019-nCoV) belonging to family Coronaviridae (Orthomyxoviridae), the virus particle is spherical or elliptical, has polymorphism, has a diameter of 60nm-140nm, is an enveloped single-stranded positive-strand RNA virus, has a genome of about 30kb, and has non-coding regions at both ends and a non-structural protein coding region and a structural protein coding region in the middle. The non-structural protein coding region mainly includes ORF1a and ORF1b genes, and the structural protein coding region mainly encodes spike protein, envelope protein, membrane protein and nucleocapsid protein. There are 7 kinds of currently known coronaviruses that can infect humans, of which 2019 novel coronaviruses (2019-nCoV) cause novel coronavirus pneumonia, SARS-CoV causes severe acute respiratory syndrome, and MERS-CoV causes middle east respiratory syndrome. 2019 novel coronavirus (2019-nCoV) has very high antigen variability and is in pandemic worldwide.
Due to their high specificity and sensitivity, molecular diagnostic techniques are widely used for the detection of viruses, particularly in clinical laboratories. Since viral RNA is present in minute amounts in clinical samples. Therefore, in many molecular diagnostic assays, extraction, amplification and analysis of viral nucleic acids are often essential, and in the molecular detection process, the design of primers plays an important role in the specificity and sensitivity of detection. The Polymerase Chain Reaction (PCR) is a common method for molecular amplification, but the PCR has high requirements on hardware devices, expensive instruments and slow amplification speed, so loop-mediated isothermal amplification (LAMP) is currently available. The loop-mediated isothermal amplification technology is a novel nucleic acid amplification technology, has the characteristics of rapidness, simplicity and strong specificity, and can hopefully replace the traditional PCR method to detect microorganisms. Therefore, aiming at a specific gene sequence of the nucleic acid of the 2019 novel coronavirus (2019-nCoV), the invention discloses a kit which can quickly and specifically detect the 2019 novel coronavirus (2019-nCoV).
Disclosure of Invention
The invention provides a kit for rapidly and specifically detecting 2019 novel coronavirus (2019-nCoV) nucleic acid and a using method thereof, which can rapidly and accurately detect the 2019 novel coronavirus (2019-nCoV) in a sample such as an oropharynx swab/nasopharynx swab and the like of a patient in real time.
The technical scheme for solving the technical problems is as follows: a kit for rapidly and specifically detecting 2019 novel coronavirus (2019-nCoV) nucleic acid comprises a working standard substance, a constant-temperature amplification reaction solution and a negative control substance,
the working standard comprises a positive control plasmid of a specific conserved sequence of a 2019 novel coronavirus (2019-nCoV), and the specific conserved sequence is SEQ ID No. 1.
The isothermal amplification reaction solution comprises 6 primers: an outer primer F3, the sequence of which is SEQ ID No. 2; an outer primer B3, the sequence of which is SEQ ID No. 3; the sequence of the inner primer FIP is SEQ ID No. 4; the sequence of the inner primer BIP is SEQ ID No. 5; the sequence of the loop primer LB is SEQ ID No. 6; and the sequence of the loop primer LF is SEQ ID No. 7.
The negative control substance is a nucleic acid extracting solution of a nasopharyngeal swab of a healthy person.
On the basis of the above technical solutions, the present invention may further specifically select as follows.
Specifically, the positive control plasmid is constructed by cloning a synthetic fragment corresponding to a 2019 novel coronavirus (2019-nCoV) gene specific conserved sequence into a pGEM-T vector, and the concentration of the synthetic fragment is 104copies/μL。
Specifically, the solvent of the isothermal amplification reaction solution is double distilled water, and the isothermal amplification reaction solution contains solutes with the following concentrations: bst DNA polymerase 8U/20. mu.L; 1U/20. mu.L AMV reverse transcriptase; 8mM MgSO4；2.4mM each dNTPs；20mM KCl；20mM(NH4)2SO4(ii) a 40Mm Tris-HCl; 0.2 wt% TritonX-100; 5pmol of outer primer F3; 5pmol outer primer B3; 40pmol of inner primer FIP; 40pmol of inner primer BIP; 20pmol of loop primer LB; 20pmol of loop primer LB; 1.25 × EvaGreen fluorescent dye.
In addition, the present invention provides a method using the above-mentioned kit for rapid and specific detection of 2019 novel coronavirus (2019-nCoV) nucleic acid, which comprises the steps of:
s1, preparing total nucleic acid of an oropharynx/nasopharynx swab sample to be detected;
s2, taking three reaction tubes, adding 20 mu L of constant-temperature amplification reaction liquid into each reaction tube, then adding 5 mu L of to-be-detected sample into the first reaction tube, adding 5 mu L of working standard substance into the second reaction tube, adding 5 mu L of negative control substance into the third reaction tube, and then placing the three reaction tubes under a constant-temperature condition for amplification reaction;
and S3, measuring a fluorescence value in real time in the amplification reaction process, and determining whether the sample to be detected contains the 2019 novel coronavirus (2019-nCoV) according to an amplification curve after the reaction is finished.
Specifically, the reaction temperature of the amplification reaction was 65 ℃ and the reaction time was 30 min.
Compared with the prior art, the invention has the beneficial effects that:
the invention screens out a specific conserved sequence containing 700bp by comparing and analyzing the gene sequence of a 2019 novel coronavirus (2019-nCoV), then designs 6 specific primers aiming at the specific conserved sequence, the 6 specific primers, Bst DNA polymerase and EvaGreen fluorescent dye (turbidity can bring errors for detection through naked eye observation, the detection sensitivity is not high, so the fluorescent dye, necessary inorganic salt and buffer solution are added to form a constant temperature amplification reaction solution, the nucleic acid of the 2019 novel coronavirus (2019-nCoV) can be rapidly amplified in the constant temperature amplification reaction solution, the rapid detection of the 2019 novel coronavirus (2019-nCoV) is realized through fluorescent real-time monitoring, and the method can be applied to the rapid detection of the 2019 novel coronavirus (2019-nCoV) in a sputum or oropharynx/nasopharynx swab sample, the operation is convenient, and the detection is convenient; the constant-temperature amplification reaction solution, the working standard substance containing the 2019 novel coronavirus (2019-nCoV) specific conserved sequence and the negative reference substance form a kit for quickly and specifically detecting the 2019 novel coronavirus (2019-nCoV), when the kit is used, 5 mu L of total nucleic acid, the working standard substance and the negative reference substance of a sample to be detected are respectively added into three reaction tubes filled with 20 mu L of the constant-temperature amplification reaction solution, and in the amplification reaction process, the fluorescence values monitored in real time are compared, so that the detection can be quickly finished.
The kit provided by the invention has very good sensitivity and specificity: the kit is matched with a fluorescence detection device, the minimum detection limit is 100copies/mL, and no amplification curve exists when nucleic acid is extracted from common respiratory pathogenic pathogens including influenza A virus, streptococcus pneumoniae, haemophilus influenzae, mycoplasma pneumoniae, mycobacterium tuberculosis, staphylococcus aureus and the like and then the detection is carried out. The kit has the advantages of high speed, high sensitivity, simple operation, low cost and the like, and is suitable for quick detection, field detection and popularization and application in basic medical institutions.
Brief description of the drawings
FIG. 1 is an amplification curve of a specificity test performed on a kit provided by the present invention;
FIG. 2 is the results of a sensitivity test performed on the kit provided by the present invention;
FIG. 3 is an amplification curve of a clinical sample experiment performed on a kit provided by the present invention;
FIG. 4 is a repetitive experiment performed by different operators and different detection platforms for the kit provided by the present invention;
Detailed Description
The technical solutions of the present invention are further described in detail with reference to the drawings and the specific embodiments, which are only used for explaining the present invention and are not used for limiting the scope of the present invention.
A kit for rapidly and specifically detecting 2019 novel coronavirus (2019-nCoV) nucleic acid comprises a working standard substance, a constant-temperature amplification reaction solution and a negative reference substance, wherein the working standard substance comprises a positive reference plasmid of a specific conserved gene sequence of the 2019 novel coronavirus (2019-nCoV), and the specific conserved sequence is SEQ ID No. 1; the isothermal amplification reaction solution comprises 6 primers: an outer primer F3, the sequence of which is SEQ ID No. 2; an outer primer B3, the sequence of which is SEQ ID No. 3; the sequence of the inner primer FIP is SEQ ID No. 4; the sequence of the inner primer BIP is SEQ ID No. 5; the sequence of the loop primer LB is SEQ ID No. 6; and the sequence of the loop primer LF is SEQ ID No. 7. The negative control substance is a nucleic acid extracting solution of a nasopharyngeal swab of a healthy person.
1. The preparation method of the working standard product comprises the following specific steps:
(1) a700 bp conserved sequence (SEQ ID No.1) of a 2019 novel coronavirus (2019-nCoV) gene is synthesized by a chemical synthesis method.
(2) The synthesized fragment was cloned into pGEM-T vector, and a 2019 novel coronavirus (2019-nCoV) positive control plasmid was constructed.
(3) The obtained positive control plasmid was transformed into E.coli DH 5. alpha. and mass-cultured in LB medium containing 1% ampicillin at 37 ℃ and then extracted with a high purity plasmid Large-Scale kit (DP116) from Tiangen Biochemical technology, Inc. (Beijing).
(4) Plasmid was purified and quantified, and diluted to 104And (5) copies/mu L to obtain the working standard substance.
2. The isothermal amplification reaction solution specifically consists of:
the solvent is double distilled water without nuclease, and the inner solutes of the double distilled water are as follows: 8U/20. mu.L Bst DNA polymerase (purchased from NEW ENGLAND BioLabs, cat # M0275), 1U/20. mu.L AMV reverse transcriptase, 8mM MgSO4、2.4mM each dNTPs、40mM Tris-HCl、20mM KCl、20mM(NH4)2SO40.2% Triton X-100, 5pmol outer primer F3, 5pmol outer primer B3, 40pmol inner primer FIP, 40pmol inner primer BIP, 20pmol loop primer LB, 1.25 × EvaGreen fluorescent dye (Biotium, cat. No. 31000).
3. The negative control product is a nucleic acid extracting solution of a nasopharyngeal swab of a healthy person, and the extracting method comprises the following steps:
A. 200 μ L of normal human nasopharyngeal swab stock solution was taken, and total nucleic acid was extracted according to the instructions of nasopharyngeal swab total nucleic acid extraction kit (DP318) of Tiangen Biochemical technology (Beijing) Ltd.
4. Preparation of oropharynx/nasopharynx swab sample to be detected
Extracting with possible 2019 novel coronavirus (2019-nCoV) carrier oropharynx/nasopharynx swab sample, and preparing the sample to be tested with the same method as the above-mentioned healthy person nasopharynx swab nucleic acid extract.
5. Reaction system preparation and amplification reaction
At least three reaction tubes: adding 20 mu L of constant-temperature amplification reaction liquid and 5 mu L of nucleic acid of a sample to be detected into a first reaction tube; adding 20 mu L of constant temperature amplification reaction liquid and 5 mu L of working standard substance into a second reaction tube; mu.L of isothermal amplification reaction solution and 5. mu.L of negative control were added to the third reaction tube.
And (3) amplification reaction: and (3) reacting the reaction tube for 30min at 65 ℃, detecting a real-time test fluorescence value, and judging whether the sample to be detected carries the 2019 novel coronavirus (2019-nCoV) nucleic acid or not according to an amplification curve.
6. Detection of
A. Specificity test
The kit is used for detecting the nucleic acid extracted from common respiratory pathogenic pathogens such as influenza A virus, streptococcus pneumoniae, haemophilus influenzae, mycoplasma pneumoniae, mycobacterium tuberculosis, staphylococcus aureus and the like. The result is shown in fig. 1, only the 2019 novel coronavirus (2019-nCoV) has an amplification curve in a specificity test experiment, and other common pathogenic pathogens have no amplification curve, which indicates that the kit provided by the invention has better specificity.
B. Sensitivity test
To a concentration of 104The standards (working standards) of the novel coronaviruses (2019-nCoV) 2019 are respectively 103、10210 and 1 copies/mu L gradient dilution, and then respectively used as total nucleic acid of a sample to be detected, and the kit is applied to detect the total nucleic acid. As shown in FIG. 2, it can be seen that the kit can detect at least 1 copy/. mu.L copies with high sensitivity.
Detection of clinical specimen of C.2019 novel coronavirus (2019-nCoV)
Samples with 6 cases of 2019 novel coronaviruses (2019-nCoV) identified to be positive in a certain Beijing hospital are collected, total nucleic acid is extracted according to the method for preparing the oropharynx/nasopharynx swab sample, and then the kit is used for detection. The results are shown in fig. 3, and 6 clinical cases were tested, 6 of which were 2019 new coronavirus (2019-nCoV) positive, and the comprehensive positive detection rate was 100%.
Repetitive detection of different operators of 2019 novel coronavirus (2019-nCoV) samples
The reproducibility and the inter-operator reproducibility were measured by eight replicates of three samples using three different operators (table 1). The operator used the same RNA extraction for each sample. The duplicate detection rate for each sample was 100%.
Repetitive detection of different detection platforms of e.2019 novel coronavirus (2019-nCoV) samples reproducibility between platforms was measured by running eight replicates of three samples on two platforms (table 2), each sample across both platforms using the same RNA extraction. The duplicate detection rate for each sample was 100%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. A kit for rapidly and specifically detecting 2019 novel coronavirus (2019-nCoV) nucleic acid is characterized by comprising a working standard substance, a constant-temperature amplification reaction solution and a negative control substance.
The working standard substance is a positive control plasmid comprising a 2019 novel coronavirus (2019-nCoV) gene specific conserved sequence, and the specific conserved sequence is SEQ ID No. 1.
The isothermal amplification reaction solution comprises 6 primers: an outer primer F3, the sequence of which is SEQ ID No. 2; an outer primer B3, the sequence of which is SEQ ID No. 3; the sequence of the inner primer FIP is SEQ ID No. 4; the sequence of the inner primer BIP is SEQ ID No. 5; the sequence of the loop primer LB is SEQ ID No. 6; and the sequence of the loop primer LF is SEQ ID No. 7.
The negative control substance is a nucleic acid extracting solution of a nasopharyngeal swab of a healthy person.
2. The kit for rapidly and specifically detecting 2019 novel coronavirus (2019-nCoV) nucleic acid as claimed in claim 1, wherein the positive control plasmid is constructed by cloning a synthetic fragment corresponding to a specific conserved sequence into a pGEM-T vector, and the concentration of the synthetic fragment is 104copies/μL。
3. The kit for rapidly and specifically detecting 2019 novel coronavirus (2019-nCoV) nucleic acid as claimed in claim 1, wherein a solvent of the isothermal amplification reaction solution is double distilled water, and the isothermal amplification reaction solution contains the following solutes in concentration: bst DNA polymerase 8U/20. mu.L; 1U/20. mu.L AMV reverse transcriptase; 8mM MgSO4；2.4mM each dNTPs；20mM KCl；20mM(NH4)2SO4(ii) a 40Mm Tris-HCl; 0.2 wt% TritonX-100; 5pmol of outer primer F3; 5pmol outer primer B3; 40pmol of inner primer FIP; 40pmol of inner primer BIP; 20pmol of loop primer LB; 20pmol of loop primer LB; 1.25 × EvaGreen fluorescent dye.
4. A method of using the kit for rapid, specific detection of 2019 novel coronavirus (2019-nCoV) nucleic acid as claimed in any one of claims 1 to 3, comprising the steps of:
s1, extracting total nucleic acid from an oropharynx/nasopharynx swab sample to be detected;
s2, taking three reaction tubes, adding 20 mu L of constant-temperature amplification reaction liquid into each reaction tube, then adding 5 mu L of sample nucleic acid to be detected into the first reaction tube, adding 5 mu L of working standard substance into the second reaction tube, adding 5 mu L of negative control substance into the third reaction tube, and then placing the three reaction tubes into a constant-temperature amplification instrument for amplification reaction;
and S3, measuring the fluorescence values of the reaction liquid in the three reaction reverse tubes in real time in the amplification reaction process, and analyzing the result after the reaction is finished to determine whether the oropharynx/nasopharynx swab sample to be measured contains 2019 novel coronavirus (2019-nCoV).
5. The method of claim 4, wherein the amplification reaction is carried out at 65 ℃ for 30min using the kit for rapid and specific detection of 2019 novel coronavirus (2019-nCoV) nucleic acid.
6. The method of claim 4, wherein the amplification reaction is followed by a melting curve assay, which can be used to determine whether the product is relatively specific.
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