CN112111597A - Fluorescence PCR kit for rapidly detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction - Google Patents
Fluorescence PCR kit for rapidly detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction Download PDFInfo
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Abstract
The invention provides a kit for rapidly detecting novel coronavirus without nucleic acid extraction, which comprises: direct RT-PCR reaction liquid, enzyme mixed liquid, negative control and positive control. The invention adds an oligonucleotide probe Block, effectively blocks the influence of coronavirus SARSr-CoV with higher homology on the detection accuracy, and ensures that the detection accuracy is higher.
Description
Technical Field
The invention relates to a fluorescence PCR kit for quickly detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction, belonging to the field of biological detection.
Background
Coronaviruses (CoV) are non-truncated single-stranded positive-strand RNA viruses belonging to the subfamily Coronaviridae (Coronaviridae) of the family Coronaviridae (Coronaviridae), and novel Coronaviruses belonging to the genus β, are enveloped, have a circular or elliptical particle shape, are usually polymorphic, and have a diameter of 60 to 140 nm. The gene characteristics of the mutant are obviously different from those of SARSr-CoV and MERSR-CoV. The present research shows that the homology with bat SARS-like coronavirus (bat-SL-CoVZC45) reaches more than 85%. Its clinical manifestations include fever, fatigue and dry cough. A few patients have nasal obstruction, watery nasal discharge, diarrhea, etc. In severe cases, dyspnea occurs after one week in many cases, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, and hemorrhagic coagulation dysfunction.
At present, according to the diagnosis and treatment scheme (trial seventh edition) for pneumonia infected by the novel coronavirus, the novel coronavirus 2019-nCoV is confirmed to be one of the following etiological or serological evidences in a suspected case: 1. the real-time fluorescence RT-PCR detects the positive of the novel coronavirus nucleic acid. 2. Viral gene sequencing, high homology to known novel coronaviruses. 3. Positive serum new coronavirus specific IgM antibody and IgG antibody; the serum specific IgG antibody of the novel coronavirus is converted from negative to positive or the recovery phase is increased by 4 times or more than the acute phase. The real-time fluorescence RT-PCR detection technology combines the obvious advantages that the PCR technology has high specificity and sensitivity, is rapid and accurate, can effectively eliminate nucleic acid cross contamination by closed tube operation, has wider and wider application, and directly detects the change of a fluorescence signal in the PCR process to obtain a detection result.
The products of real-time fluorescent RT-PCR amplification are nucleic acid fragments, nucleic acids of virus coronaviruses are all wrapped in protein capsids, and the viruses exist in clinical samples with complex components, so the clinical samples need to be processed before amplification, and the nucleic acids can be amplified after being released and purified. At the present stage, nucleic acid of a sample is extracted mainly by a centrifugal column method and a magnetic bead method, the two methods are subjected to steps of cracking, combining, washing, rinsing, eluting and the like, the operation is complex, the time consumption is long, and a large amount of protein allosteric effectors and organic solvents are required. The sample extraction easily causes cross contamination among samples to cause false positive, and meanwhile, the nucleic acid extraction also needs professional laboratory equipment and environment and has special requirements on operators.
The gene comparison shows that the genome sequence of the novel coronavirus 2019-nCoV is highly homologous with the SARSr-CoV, and a real-time fluorescence RT-PCR method capable of effectively distinguishing the genome sequence and the SARSr-CoV and accurately detecting the novel coronavirus 2019-nCoV nucleic acid is urgently needed to be invented.
Therefore, in order to meet the clinical rapid detection requirement of the novel coronavirus 2019-nCoV, the invention of a direct amplification real-time fluorescent RT-PCR kit which can improve the detection accuracy and has low operation requirement is urgently needed.
Disclosure of Invention
The invention aims to provide a fluorescent PCR kit for rapidly detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction, so that the effects of improving detection accuracy and low operation requirement are achieved.
The invention adopts the following technical scheme:
the invention provides a primer and a probe for detecting real-time fluorescent RT-PCR of a novel coronavirus 2019-nCoV nucleic acid, which are characterized in that:
designing upstream and downstream primers and probes aiming at the novel coronavirus open reading frame 1a/b and the nucleocapsid protein, wherein the gene primers and probe sequences of the novel coronavirus open reading frame 1a/b and the nucleocapsid protein are as follows:
novel coronavirus open reading frame 1a/b gene upstream primer 2019nCOV (ORF) -F: GCACGTGCTGGTAAAGCTTCA
Novel coronavirus open reading frame 1a/b gene downstream primer 2019nCOV (ORF) -R: TTTAATTTCAAAAGGTGTCTGCAAT
Novel coronavirus open reading frame 1a/b gene probe 2019nCOV (ORF) -P: TTATTGACACTAAGAGGGGTGTATACTGCTGCC
Nucleocapsid protein gene upstream primer 2019nCOV (N) -F:
TGTCTGATAATGGACCCCAAAAT
nucleocapsid protein gene downstream primer 2019nCOV (N) -R:
CCGACGTTGTTTTGATCGC
nucleocapsid protein gene probe 2019ncov (n) -P:
ACCCTCAGATTCAACTGGCAGTAACCAGAA。
the invention also provides a real-time fluorescence RT-PCR method for detecting the novel coronavirus 2019-nCoV nucleic acid, which is characterized by comprising the following steps of: a probe aiming at a specific sequence for blocking the SARSr-CoV sequence is named as Block, the 3' end of the probe is subjected to phosphorylation modification, the sequence of the Block is a highly homologous part with a region to be detected of the novel coronavirus 2019-nCoV in the SARSr-CoV sequence, and the sequence of the Block is as follows:
SARS(ORF)-B：GCACGCGCGGGCAAGTCAATG-PO4
SARS(N)-B：TGTCTGATAATGGACCCCAATCG-PO4。
the invention also provides a nucleic acid extraction-free fluorescent RT-PCR kit for detecting the novel coronavirus 2019-nCoV nucleic acid, which is characterized by comprising the following components in parts by weight:
direct RT-PCR reaction liquid, enzyme mixed liquid, negative control and positive control,
the Direct RT-PCR reaction solution comprises 0.8-1.2% Tween20, 0.7-0.9% polyethylene glycol octylphenyl ether, 20mM-50mM NaOH, 0.3-0.6% PEG8000, 4-5mM magnesium chloride, 10-30mM Tris-HCl (pH 8.2), 30-50mM KCl, 10-20mM ammonium sulfate, 0.1-0.3mg/mL calf serum albumin, 200. mu.M-400. mu.M dATP, 200. mu.M-400. mu.M dGTP, 200. mu.M-400. mu.M dCTP, 100. mu.M-200. mu.M dTTP, 100. mu.M-200. mu.M dUTP, 0.1-0.3. mu.M primer (2019nCOV (ORF)) -F, 2019nCOV (ORF)) (ORF) -R, 2019nCOV (N), (2019 nCOV) (R, HBB-F, HBB-R), 0.1-0.2M ORF-2 nCOV (ORF) -R), 2019nCOV (N) -P, HBB-P), 0.3-0.5. mu.M Block (SARS (ORF) -B, SARS (N) -B) and DEPC water.
Further, the fluorescence RT-PCR kit for detecting the novel coronavirus 2019-nCoV nucleic acid, which is free of nucleic acid extraction, also has the following characteristics: the enzyme mixed solution comprises 2-5U/mu L of hot start Taq enzyme, 20-40U/mu L of reverse transcriptase, 1-2U/mu L of heat-sensitive UDG enzyme and 25-40U/mu L of RNase inhibitor.
Further, the fluorescence RT-PCR kit for detecting the novel coronavirus 2019-nCoV nucleic acid, which is free of nucleic acid extraction, also has the following characteristics:
the negative control was sterilized saline.
Further, the fluorescence RT-PCR kit for detecting the novel coronavirus 2019-nCoV nucleic acid, which is free of nucleic acid extraction, also has the following characteristics:
the positive control is 103copy/uL of novel coronavirus open reading frame 1a/b gene and nucleocapsid protein group in nucleic acid containing novel coronavirus 2019-nCoVPseudoviruses with an internal standard of the RNA sequence of the beta globin gene.
Compared with the existing novel coronavirus 2019-nCoV nucleic acid detection kit, the kit has the following advantages:
(1) the invention adds an oligonucleotide probe Block, effectively blocks the influence of coronavirus SARSr-CoV with higher homology on the detection accuracy, and ensures that the detection accuracy is higher.
(2) The invention optimizes the real-time RT-PCR detection process, avoids nucleic acid extraction and can obviously improve the RT-PCR detection efficiency. Reduces the RT-PCR detection cost and has higher economic benefit.
(3) The invention provides a direct amplification real-time fluorescent RT-PCR kit with high accuracy and low operation requirement, and the kit can meet the requirement of the market on the detection of the nucleic acid of the novel coronavirus 2019-nCoV.
Drawings
Fig. 1A and 1B are schematic diagrams of Block operation.
FIG. 2 shows the amplification curve of the quality control of the kit, wherein FIG. 2A shows the amplification curve of a positive control in the RT-PCR reaction solution, FIG. 2B shows the amplification curve of a negative control in the RT-PCR reaction solution, and FIG. 2C shows the amplification curve of an internal control beta globin gene in the RT-PCR reaction solution, wherein the abscissa is the PCR cycle number and the ordinate is the fluorescence value.
FIG. 3 shows the amplification curve of the new coronavirus 2019-nCoV positive sample after extraction in RT-PCR reaction solution and the amplification curve of sample loading without extraction, wherein the abscissa is the PCR cycle number and the ordinate is the fluorescence value. Wherein, FIG. 3A is a detection curve of the detection of the internal standard gene after extraction by the magnetic bead method and the hands-free direct detection of the present invention. FIG. 3B is a detection curve of the 2019n-CoV ORFlab gene after extraction by the magnetic bead method and the hands-free direct detection of the invention. FIG. 3C is a detection curve of 2019N-CoV virus N gene after extraction using the magnetic bead method and the hands-free direct detection of the present invention.
FIG. 4 shows the amplification curve of the new coronavirus 2019-nCoV negative sample after extraction in RT-PCR reaction solution and the amplification curve of sample loading without extraction, wherein the abscissa is the PCR cycle number and the ordinate is the fluorescence value. Wherein FIG. 4A is a detection curve of detection of an internal standard gene after extraction using a magnetic bead method and hands-free direct detection of the present invention. FIG. 4B is a detection curve of the 2019n-CoV ORFlab gene after extraction by the magnetic bead method and the hands-free direct detection of the present invention. FIG. 4C is a detection curve of 2019N-CoV virus N gene after extraction using the magnetic bead method and the hands-free direct detection of the present invention.
FIG. 5 shows samples with Block added to detect SARSr-CoV positive and new coronavirus 2019n-CoV negative and samples without Block added to detect SARSr-CoV positive and new coronavirus 2019n-CoV negative. FIG. 5A is a graph showing the detection of the internal standard gene after addition of Block and the detection without addition of Block. FIG. 5B is a graph showing the detection of the ORFlab gene of 2019n-CoV virus after addition of Block and the detection without addition of Block. FIG. 5C is a graph showing the detection of the 2019N-CoV virus N gene after addition of Block and the detection without addition of Block.
Detailed Description
The technical solution of the present invention is further described below with reference to specific examples, but the scope of the present invention is not limited thereto.
The first embodiment is as follows: composition of novel coronavirus 2019-nCoV nucleic acid detection kit
The composition of the novel coronavirus 2019-nCoV nucleic acid detection kit is shown in Table 1.
Table 1: novel coronavirus 2019-nCoV nucleic acid detection kit composition
Wherein the final concentration of each component of the Direct RT-PCR reaction solution is 1% Tween20, 0.8% polyethylene glycol octylphenyl ether, 50mM NaOH, 0.5% PEG8000, 4mM magnesium chloride, 20mM Tris-HCl (pH 8.2), 40mM KCl, 20mM ammonium sulfate, 0.3mg/mL calf serum albumin, 200. mu.M dATP, 200. mu.M dGTP, 200. mu.M dCTP, 100. mu.M dTTP, 100. mu.M dUTP, 0.2. mu.M primer (2019nCOV (ORF) -F, 2019nCOV (ORF) -R, 2019nCOV (N) -F, 2019nCOV (N) -R, HBB-F, HBB-R), 0.1. mu.M probe (2019nCOV (ORF) -P, 2019nCOV (N) -P, HBB-P), 0.4. mu.M Block (DEN) -B, SARS-P, and water.
The principle of Block is shown in fig. 1A and 1B: block functions to Block the SARSr-CoV sequence, organizing its reverse transcription. A probe of a specific sequence playing a role in blocking is named as Block, the 3' end of the probe is subjected to phosphorylation modification, the sequence of the Block is directed at a part which is highly homologous with a region to be detected of the novel coronavirus 2019-nCoV in a SARSr-CoV sequence, and the sequence of the Block is as follows:
SARS(ORF)-B：GCACGCGCGGGCAAGTCAATG-PO4
SARS(N)-B：TGTCTGATAATGGACCCCAATCG-PO4。
the enzyme mixture was 3U/. mu.L of hot start Taq enzyme, 30U/. mu.L reverse transcriptase, 1.5U/. mu.L of heat-sensitive UDG enzyme, and 36U/. mu.L of RNase inhibitor.
Wherein, the negative control is sterilized normal saline.
Wherein the positive control is 103copy/uL of pseudovirus containing the RNA sequence of ORF1ab gene, N gene and internal standard beta globin gene in the novel coronavirus 2019-nCoV nucleic acid, and sterilized normal saline.
FIG. 2 shows the amplification curve of the quality control of the kit, wherein FIG. 2A shows the amplification curve of a positive control in the RT-PCR reaction solution, FIG. 2B shows the amplification curve of a negative control in the RT-PCR reaction solution, and FIG. 2C shows the amplification curve of an internal control beta globin gene in the RT-PCR reaction solution, wherein the abscissa is the PCR cycle number and the ordinate is the fluorescence value.
Example two: application of novel coronavirus 2019-nCoV nucleic acid detection kit
(1) Direct amplification RT-PCR detection
Taking the Direct RT-PCR reaction solution and the enzyme mixed solution (23.5 mu L/person of the Direct RT-PCR reaction solution and 1.5 mu L/person of the enzyme mixed solution) according to the proportion, fully mixing the reaction solution and the enzyme mixed solution, and subpackaging the mixture into PCR reaction tubes according to 25 mu L/tube for later use. 5 μ L of nasopharyngeal swab/sputum/bronchial lavage/alveolar lavage is added to the prepared RT-PCR reaction solution, and the covering tube is tightly covered with bubbles, which can be flicked with fingers to remove the bubbles. Centrifuge at 5000 rpm for 30 seconds until no liquid beads were evident on the tube wall.
(2) Direct amplification of RT-PCR
And placing the PCR reaction tube into a sample groove of an amplification instrument, setting a positive control, a negative control and a sample to be detected in a corresponding sequence, and setting the name of the sample.
The fluorescence detection channel selects FAM/CY 5/ROX. The PCR reaction cycle parameters were set as shown in table 2 below.
Table 2: PCR reaction parameters
(3) Analysis of results
And automatically storing results after the reaction is finished, and analyzing the amplification curves of the detection target and the reference gene respectively. Adjusting the Start value, the End value and the Threshold value of Baseline according to the analyzed images, wherein the Start value is 3-15, the End value is 5-20, adjusting the amplification curve of the negative control to be flat or lower than a Threshold line, and recording the line type and the CT value of FAM/CY5/ROX respectively.
Quality control: negative control: the FAM, ROX and internal standard (CY5) channels have no Ct value or Ct > 38. Positive control: the Ct of FAM, ROX and internal standard (CY5) channels is less than or equal to 38. The above requirements need to be met simultaneously in the same experiment, otherwise, the experiment is invalid and needs to be performed again.
And (5) judging a result:
whether the internal standard (CY5) has a typical S-type amplification curve is analyzed firstly, and if so, the Ct is less than or equal to 40, which indicates that the detection is effective, and the subsequent analysis can be continued.
(1) If the FAM channel detects a typical S-type amplification curve and the Ct is less than or equal to 40, the 2019-nCoV virus ORF1ab gene is positive. If the typical S-type amplification curve is not detected in the FAM channel, the ORF1ab gene of the 2019-nCoV virus is negative.
(2) If a typical S-type amplification curve is detected by the ROX channel and Ct is less than or equal to 40, the 2019-nCoV virus N gene is positive. If the ROX channel does not detect a typical S-type amplification curve, it indicates that the N gene of the 2019-nCoV virus is negative.
(3) If the internal standard does not detect Ct or Ct is more than 40 in the CY5 channel, the result indicates that the concentration of the detected sample is too low or the interferent inhibits the reaction, and the experiment needs to be prepared again.
(4) For positive samples, the internal standard detection result does not make requirements; and for a negative sample, detecting the internal standard to be positive, if the internal standard is detected to be negative, detecting the result of the sample to be invalid, searching and eliminating the reason, resampling the sample, and performing repeated tests.
(5) The 2019-nCoV virus was judged to be positive only when both the 2019-nCoV virus ORF1ab gene and the 2019-nCoV virus N gene were positive.
FIG. 3 shows the amplification curve of the new coronavirus 2019-nCoV positive sample after extraction in RT-PCR reaction solution and the amplification curve of sample loading without extraction, wherein the abscissa is the PCR cycle number and the ordinate is the fluorescence value. Wherein, FIG. 3A is a detection curve of the detection of the internal standard gene after extraction by the magnetic bead method and the hands-free direct detection of the present invention. FIG. 3B is a detection curve of the 2019n-CoV ORFlab gene after extraction by the magnetic bead method and the hands-free direct detection of the invention. FIG. 3C is a detection curve of 2019N-CoV virus N gene after extraction using the magnetic bead method and the hands-free direct detection of the present invention.
FIG. 4 shows the amplification curve of the new coronavirus 2019-nCoV negative sample after extraction in RT-PCR reaction solution and the amplification curve of sample loading without extraction, wherein the abscissa is the PCR cycle number and the ordinate is the fluorescence value. Wherein FIG. 4A is a detection curve of detection of an internal standard gene after extraction using a magnetic bead method and hands-free direct detection of the present invention. FIG. 4B is a detection curve of the 2019n-CoV ORFlab gene after extraction by the magnetic bead method and the hands-free direct detection of the present invention. FIG. 4C is a detection curve of 2019N-CoV virus N gene after extraction using the magnetic bead method and the hands-free direct detection of the present invention.
FIG. 5 shows samples with Block added to detect SARSr-CoV positive and new coronavirus 2019n-CoV negative and samples without Block added to detect SARSr-CoV positive and new coronavirus 2019n-CoV negative. FIG. 5A is a graph showing the detection of the internal standard gene after addition of Block and the detection without addition of Block. FIG. 5B is a graph showing the detection of the ORFlab gene of 2019n-CoV virus after addition of Block and the detection without addition of Block. FIG. 5C is a graph showing the detection of the 2019N-CoV virus N gene after addition of Block and the detection without addition of Block.
Sequence listing
<110> Anhui Coincident Biotechnology Co., Ltd
<120> fluorescence PCR kit for rapidly detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction
<130> JSP12001134
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial
<220>
Claims (6)
1. A primer and a probe for real-time fluorescent RT-PCR for detecting novel coronavirus 2019-nCoV nucleic acid are characterized in that:
designing upstream and downstream primers and probes aiming at the novel coronavirus open reading frame 1a/b and the nucleocapsid protein, wherein the gene primers and probe sequences of the novel coronavirus open reading frame 1a/b and the nucleocapsid protein are as follows:
novel coronavirus open reading frame 1a/b gene upstream primer 2019nCOV (ORF) -F: GCACGTGCTGGTAAAGCTTCA
Novel coronavirus open reading frame 1a/b gene downstream primer 2019nCOV (ORF) -R: TTTAATTTCAAAAGGTGTCTGCAAT
Novel coronavirus open reading frame 1a/b gene probe 2019nCOV (ORF) -P: TTATTGACACTAAGAGGGGTGTA
TACTGCTGCC
Nucleocapsid protein gene upstream primer 2019nCOV (N) -F:
TGTCTGATAATGGACCCCAAAAT
nucleocapsid protein gene downstream primer 2019nCOV (N) -R:
CCGACGTTGTTTTGATCGC
nucleocapsid protein gene probe 2019ncov (n) -P:
ACCCTCAGATTCAACTGGCAGTAACCAGAA。
2. a real-time fluorescent RT-PCR method for detecting novel coronavirus 2019-nCoV nucleic acid, which is characterized by comprising the following steps: a probe aiming at a specific sequence for blocking the SARSr-CoV sequence is named as Block, the 3' end of the probe is subjected to phosphorylation modification, and the sequence of the Block is as follows:
SARS(ORF)-B：GCACGCGCGGGCAAGTCAATG-PO4
SARS(N)-B：TGTCTGATAATGGACCCCAATCG-PO4。
3. a fluorescence RT-PCR kit for detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction is characterized by comprising:
direct RT-PCR reaction liquid, enzyme mixed liquid, negative control and positive control,
the Direct RT-PCR reaction solution comprises 0.8-1.2% Tween20, 0.7-0.9% polyethylene glycol octylphenyl ether, 20mM-50mM NaOH, 0.3-0.6% PEG8000, 4-5mM magnesium chloride, 10-30mM Tris-HCl (pH 8.2), 30-50mM KCl, 10-20mM ammonium sulfate, 0.1-0.3mg/mL calf serum albumin, 200. mu.M-400. mu.M dATP, 200. mu.M-400. mu.M dGTP, 200. mu.M-400. mu.M dCTP, 100. mu.M-200. mu.M dTTP, 100. mu.M-200. mu.M dUTP, 0.1-0.3. mu.M primer (2019nCOV (ORF)) -F, 2019nCOV (ORF)) (ORF) -R, 2019nCOV (N), (2019 nCOV) (R, HBB-F, HBB-R), 0.1-0.2M ORF-2 nCOV (ORF) -R), 2019nCOV (N) -P, HBB-P), 0.3-0.5. mu.M Block (SARS (ORF) -B, SARS (N) -B) and DEPC water.
4. The nucleic acid extraction-free fluorescent RT-PCR kit for detecting the novel coronavirus 2019-nCoV nucleic acid as claimed in claim 3, wherein the kit comprises:
the enzyme mixed solution comprises 2-5U/mu L of hot start Taq enzyme, 20-40U/mu L of reverse transcriptase, 1-2U/mu L of heat-sensitive UDG enzyme and 25-40U/mu L of RNase inhibitor.
5. The nucleic acid extraction-free fluorescent RT-PCR kit for detecting the novel coronavirus 2019-nCoV nucleic acid as claimed in claim 3, wherein the kit comprises:
the negative control was sterilized saline.
6. The nucleic acid extraction-free fluorescent RT-PCR kit for detecting the novel coronavirus 2019-nCoV nucleic acid as claimed in claim 3, wherein the kit comprises:
the positive control is 103A pseudovirus containing the RNA sequence of the novel coronavirus open reading frame 1a/b gene, the nucleocapsid protein gene and the internal standard beta globin gene in the novel coronavirus 2019-nCoV nucleic acid in a copy/uL mode.
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| CN113151583A (en) * | 2020-12-29 | 2021-07-23 | 苏州新波生物技术有限公司 | Detection kit capable of directly amplifying sample containing SARS CoV-2 virus and detection method thereof |
| WO2022194756A3 (en) * | 2021-03-15 | 2022-12-01 | F. Hoffmann-La Roche Ag | Compositions and methods for detecting severe acute respiratory syndrome coronavirus 2 (sars-cov-2) variants having spike protein mutations |
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