CN112111597B - Fluorescent PCR kit for rapidly detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction - Google Patents
Fluorescent 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 coronaviruses without nucleic acid extraction, which comprises the following components: direct RT-PCR reaction solution, enzyme mixed solution, negative control and positive control. The invention increases the influence of oligonucleotide probe Block on the detection accuracy by effectively blocking coronavirus SARSr-CoV with higher homology, so that the detection accuracy is higher.
Description
Technical Field
The invention relates to a fluorescent PCR kit for rapidly detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction, belonging to the field of biological detection.
Background
Coronaviruses (CoV) are single-stranded positive strand RNA viruses that are not split, belonging to the Coronaviridae subfamily (Coronaviridae) and novel Coronaviruses belonging to the genus β, enveloped, circular or oval, often polymorphic, and 60-140nm in diameter. The gene characteristics are obviously different from SARSr-CoV and MERSr-CoV. The current research shows that the homology with bat SARS-like coronavirus (bat-SL-CoVZC 45) is more than 85%. The clinical manifestations of the traditional Chinese medicine are fever, hypodynamia and dry cough. Few patients have symptoms such as nasal obstruction, watery nasal discharge, diarrhea, etc. The severe cases often develop dyspnea after one week, and the severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, and clotting dysfunction.
The current diagnosis of novel coronavirus 2019-nCoV according to the diagnosis and treatment protocol for pneumonia of novel coronavirus infection (seventh edition of trial) is one of the following etiologies or serological evidence for suspected cases: 1. and detecting the positive of the novel coronavirus nucleic acid by real-time fluorescence RT-PCR. 2. Viral gene sequencing is highly homologous to known novel coronaviruses. 3. Serum novel coronavirus specific IgM antibodies and IgG antibodies positive; the novel serum coronavirus specific IgG antibodies are changed from negative to positive or the recovery period is increased by 4 times or more than the acute period. The real-time fluorescent RT-PCR detection technology integrates the obvious advantages of high specificity, high sensitivity, rapidness and accuracy and capability of effectively eliminating the cross contamination of nucleic acid by the closed-tube operation, and has wider and wider application, and the change of fluorescent signals in the PCR process is directly detected to obtain a detection result.
The real-time fluorescent RT-PCR amplified product is a nucleic acid fragment, the nucleic acid of the virus coronavirus is wrapped in a protein capsid, and the virus exists in a clinical sample with complex components, so that the clinical sample needs to be treated before the amplification, and the nucleic acid can be amplified after being released and purified. The nucleic acid of the sample is extracted at the present stage mainly by adopting a centrifugal column method and a magnetic bead method, and the two methods are subjected to steps of cracking, combining, washing, rinsing, eluting and the like, so that the operation is complex, the time consumption is long, and a large amount of protein allosteric hindrance agent and organic solvent are required. Sample extraction is easy to cause cross contamination among samples, causes false positive, and meanwhile, the nucleic acid extraction also needs professional laboratory equipment and environment, and has special requirements for operators.
Through gene comparison, the genome sequence of the novel coronavirus 2019-nCoV is found to be highly homologous with SARSr-CoV, and a method for real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) capable of effectively distinguishing the genome sequence from the SARSr-CoV and accurately detecting the nucleic acid of the novel coronavirus 2019-nCoV is urgently needed.
Therefore, in order to meet the clinical rapid detection requirement of the novel coronavirus 2019-nCoV, it is highly desirable to invent a direct amplification real-time fluorescent RT-PCR kit capable of improving the detection accuracy and having low operation requirements.
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, thereby achieving the effects of improving detection accuracy and having low operation requirements.
The invention adopts the following technical scheme:
the invention provides a primer and a probe for detecting real-time fluorescence RT-PCR of novel coronavirus 2019-nCoV nucleic acid, which are characterized in that:
upstream and downstream primers and probes are designed for the novel coronavirus open reading frame 1a/b and nucleocapsid proteins, and the gene primers and probe sequences for the novel coronavirus open reading frame 1a/b and nucleocapsid proteins 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 gene downstream primer 2019nCOV (N) -R:
CCGACGTTGTTTTGATCGC
nucleocapsid protein gene probe 2019nCOV (N) -P:
ACCCTCAGATTCAACTGGCAGTAACCAGAA。
the invention also provides a real-time fluorescent RT-PCR method for detecting novel coronavirus 2019-nCoV nucleic acid, which is characterized in that: the probe for the specific sequence of the closed 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 aimed at a part which is highly homologous to a novel coronavirus 2019-nCoV to-be-detected region in the SARSr-CoV sequence, wherein the sequence of the Block is as follows:
SARS(ORF)-B：GCACGCGCGGGCAAGTCAATG-PO 4
SARS(N)-B：TGTCTGATAATGGACCCCAATCG-PO 4 。
the invention also provides a fluorescent RT-PCR kit for detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction, which is characterized by comprising the following components:
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 μM-400 μM dATP, 200 μM-400 μM dGTP, 200 μM-400 μM dCTP, 100 μM-200 μM dTTP, 0.1-0.3 μM primers (2019 nCOV (ORF) -F, 2019nCOV (ORF) -R, 2019nCOV (N) -R, HBB-F, HBB-R), 0.1-0.2 μM probes (2019 nCOV) -62, and 50.3 μM primers (2019 nCOV (ORF) -N) -62.3 μM, and SARS (BK.3.5-6.3 μM water.
Further, the fluorescent RT-PCR kit for detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction of the invention has the following characteristics: the enzyme mixture 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 fluorescent RT-PCR kit for detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction of the invention has the following characteristics:
the negative control is sterilized normal saline.
Further, the fluorescent RT-PCR kit for detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction of the invention has the following characteristics:
the positive control was 10 3 copy/uL of pseudovirus comprising RNA sequences of the novel coronavirus open reading frame 1a/b gene, nucleocapsid protein gene and internal standard β -globin gene in the novel coronavirus 2019-nCoV nucleic acid.
Compared with the existing novel coronavirus 2019-nCoV nucleic acid detection kit, the invention has the following advantages:
(1) The invention increases the influence of oligonucleotide probe Block on the detection accuracy by effectively blocking coronavirus SARSr-CoV with higher homology, so that the detection accuracy is higher.
(2) The invention optimizes the real-time RT-PCR detection flow, avoids nucleic acid extraction, and can obviously improve the efficiency of RT-PCR detection. Reduce the cost of RT-PCR detection and have higher economic benefit.
(3) The invention provides a direct amplification real-time fluorescent RT-PCR kit with high accuracy and low operation requirement, which meets the requirements of the market on novel coronavirus 2019-nCoV nucleic acid detection.
Drawings
Fig. 1A and 1B are Block operation schematic diagrams.
FIG. 2 shows the amplification curve of the quality control of the kit, wherein FIG. 2A shows the amplification curve of the positive control in the RT-PCR reaction solution, FIG. 2B shows the amplification curve of the negative control in the RT-PCR reaction solution, and FIG. 2C shows the amplification curve of the internal control beta globin gene in the RT-PCR reaction solution, wherein the abscissa indicates the number of PCR cycles and the ordinate indicates the fluorescence value.
FIG. 3 shows the amplification curve of a novel coronavirus 2019-nCoV positive sample extracted in RT-PCR reaction solution and the amplification curve directly subjected to sample addition without extraction, wherein the abscissa represents the PCR cycle number and the ordinate represents the fluorescence value. Wherein, FIG. 3A is a detection curve 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 2019n-CoV virus ORFlab gene after extraction using the magnetic bead method and the hands-free direct detection of the present 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 a novel coronavirus 2019-nCoV negative sample extracted in the RT-PCR reaction solution and the amplification curve directly subjected to sample addition without extraction, wherein the abscissa indicates the number of PCR cycles and the ordinate indicates the fluorescence value. Wherein FIG. 4A is a detection curve of the internal standard gene after extraction using the magnetic bead method and the hands-free direct detection of the present invention. FIG. 4B is a detection curve of 2019n-CoV virus ORFlab gene after extraction using 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 that were positive for SARSr-CoV and negative for novel coronavirus 2019n-CoV with the addition of Block and samples that were positive for SARSr-CoV and negative for novel coronavirus 2019n-CoV without the addition of Block. FIG. 5A is a graph showing detection of internal standard genes after addition of Block and detection without addition of Block. FIG. 5B is a graph of detection of 2019n-CoV virus ORFlab gene after addition of Block and detection without addition of Block. FIG. 5C is a graph of detection of 2019N-CoV virus N gene after addition of Block and detection without addition of Block.
Detailed Description
The technical scheme of the present invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Embodiment one: composition of novel coronavirus 2019-nCoV nucleic acid detection kit
The compositions of the novel coronavirus 2019-nCoV nucleic acid detection kit are 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 octyl phenyl 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 (2019 nCOV (ORF) -F, 2019nCOV (ORF) -R, 2019nCOV (N) -F, 2019nCOV (N) -R, HBB-F, HBB-R), 0.1 mu M probe (2019 nCOV (ORF) -P, 2019nCOV (N) -P, HBB-P), 0.4 mu M Block (SARS (ORF) -B, SARS (N) -B) and DEPC water.
The principle of Block is shown in fig. 1A and 1B: block functions to Block the SARSr-CoV sequence, organizing its reverse transcription. The probe of the specific sequence with the blocking function is named as Block, the 3' -end of the probe is subjected to phosphorylation modification, and the sequence of the Block aims at a part which is highly homologous to a novel coronavirus 2019-nCoV to-be-detected region 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 enzyme mixture was 3U/. Mu.L of hot start Taq enzyme, 30U/. Mu.L of reverse transcriptase, 1.5U/. Mu.L of heat sensitive UDG enzyme, 36U/. Mu.L of RNase inhibitor.
Wherein the negative control is sterilized normal saline.
Wherein the positive control is 10 3 copy/uL pseudovirus containing RNA sequences of ORF1ab gene, N gene and internal standard β -globin gene in novel coronavirus 2019-nCoV nucleic acid, sterilized normal saline.
FIG. 2 shows the amplification curve of the quality control of the kit, wherein FIG. 2A shows the amplification curve of the positive control in the RT-PCR reaction solution, FIG. 2B shows the amplification curve of the negative control in the RT-PCR reaction solution, and FIG. 2C shows the amplification curve of the internal control beta globin gene in the RT-PCR reaction solution, wherein the abscissa indicates the number of PCR cycles and the ordinate indicates the fluorescence value.
Embodiment two: application of novel coronavirus 2019-nCoV nucleic acid detection kit
(1) Direct amplification RT-PCR detection
Taking Direct RT-PCR reaction liquid and enzyme mixed liquid (23.5 mu L/part of Direct RT-PCR reaction liquid and 1.5 mu L/part of enzyme mixed liquid) according to the proportion, fully mixing uniformly, and sub-packaging into PCR reaction tubes according to 25 mu L/tube for standby. 5 mu L of nasopharyngeal swab/sputum/broncholavage liquid/alveolar lavage liquid are added to the prepared RT-PCR reaction solution respectively, the tube is covered tightly, if bubbles exist, the tube can be flicked by fingers, and the bubbles are removed. Centrifuge at 5000 rpm for 30 seconds until no obvious beads are present on the tube wall.
(2) Direct amplification RT-PCR amplification
And placing the PCR reaction tube into a sample groove of an amplification instrument, setting positive control, negative control and a sample to be tested according to the corresponding sequence, and setting sample names.
The fluorescence detection channel was FAM/CY5/ROX. The PCR reaction cycle parameters were set as shown in Table 2 below.
Table 2: PCR reaction parameters
(3) Analysis of results
And after the reaction is finished, automatically storing the result, and respectively analyzing the amplification curves of the detection target and the internal reference gene. And (3) regulating the Start value, end value and Threshold value of the Baseline according to the analyzed image, setting the Start value at 3-15, the End value at 5-20, regulating the amplification curve of the negative control to be straight or lower than a Threshold line, and recording the observed line type and CT value of FAM/CY5/ROX respectively.
And (3) quality control: negative control: FAM, ROX and internal standard (CY 5) channels have no Ct value or Ct > 38. Positive control: FAM, ROX and internal standard (CY 5) channels have Ct less than or equal to 38. All the requirements are required to be met in the same experiment, otherwise, the experiment is invalid and the experiment needs to be carried out again.
And (3) judging results:
whether the internal standard (CY 5) has a typical S-shaped amplification curve or not is analyzed, if yes, ct is less than or equal to 40, the detection is effective, and the subsequent analysis can be continued.
(1) If the typical S-type amplification curve is detected by the FAM channel and Ct is less than or equal to 40, the ORF1ab gene of the 2019-nCoV virus is positive. If the FAM channel does not detect a typical S-type amplification curve, the ORF1ab gene of the 2019-nCoV virus is negative.
(2) If the ROX channel detects a typical S-type amplification curve and Ct is less than or equal to 40, the N gene of the 2019-nCoV virus is positive. If the ROX channel does not detect a typical S-type amplification curve, the 2019-nCoV virus N gene is negative.
(3) If Ct is not detected in the CY5 channel or Ct is more than 40, the concentration of the detected sample is too low or the inhibition reaction of the interferents exists, and the experiment needs to be prepared again.
(4) For positive samples, the internal standard detection result is not required; for a negative sample, the internal standard detection should be positive, if the internal standard detection is negative, the detection result of the sample is invalid, the cause should be searched and removed, and the sample is resampled for repeated tests.
(5) Only when both the ORF1ab gene of 2019-nCoV virus and the N gene of 2019-nCoV virus were positive, it was judged that 2019-nCoV virus was positive.
FIG. 3 shows the amplification curve of a novel coronavirus 2019-nCoV positive sample extracted in RT-PCR reaction solution and the amplification curve directly subjected to sample addition without extraction, wherein the abscissa represents the PCR cycle number and the ordinate represents the fluorescence value. Wherein, FIG. 3A is a detection curve 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 2019n-CoV virus ORFlab gene after extraction using the magnetic bead method and the hands-free direct detection of the present 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 a novel coronavirus 2019-nCoV negative sample extracted in the RT-PCR reaction solution and the amplification curve directly subjected to sample addition without extraction, wherein the abscissa indicates the number of PCR cycles and the ordinate indicates the fluorescence value. Wherein FIG. 4A is a detection curve of the internal standard gene after extraction using the magnetic bead method and the hands-free direct detection of the present invention. FIG. 4B is a detection curve of 2019n-CoV virus ORFlab gene after extraction using 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 that were positive for SARSr-CoV and negative for novel coronavirus 2019n-CoV with the addition of Block and samples that were positive for SARSr-CoV and negative for novel coronavirus 2019n-CoV without the addition of Block. FIG. 5A is a graph showing detection of internal standard genes after addition of Block and detection without addition of Block. FIG. 5B is a graph of detection of 2019n-CoV virus ORFlab gene after addition of Block and detection without addition of Block. FIG. 5C is a graph of detection of 2019N-CoV virus N gene after addition of Block and detection without addition of Block.
Sequence listing
<110> Anhui Cooperation Biotech Co., ltd
<120> fluorescent PCR kit for rapid detection of 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 (4)
1. A fluorescent RT-PCR kit for detecting novel coronavirus 2019-nCoV nucleic acid without nucleic acid extraction, 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 octyl phenyl 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 μM-400 μM dATP, 200 μM-400 μM dGTP, 200 μM-400 μM dCTP, 100 μM-200 μM dTTP, 0.1-0.3 μM primers (2019 nCOV (ORF) -F, 2019nCOV (ORF) -R, 2019nCOV (N) -R, HBB-F, HBB-R), 0.1-0.2 μM probes (2019 nCOV) -N, 2019nCOV (N) -62.3 μM, and SARS (BK) -2.5.3 μM primers (2019 nCOV (ORF) -N, 2019nCOV (N) -20.3 μM and water (PbR);
wherein, the novel coronavirus designs upstream and downstream primers and probes aiming at the novel coronavirus open reading frame 1a/b and nucleocapsid protein, and the sequences of the gene primers and probes of the novel coronavirus open reading frame 1a/b and nucleocapsid protein are as follows:
viral 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 gene downstream primer 2019nCOV (N) -R: CCGACGTTGTTTTGATCGC
Nucleocapsid protein gene probe 2019nCOV (N) -P: ACCCTCAGATTCAACTGGCAGTAACCAGAA;
the probe for the specific sequence of the blocked SARSr-CoV sequence was named Block, the 3' -end of which was modified by phosphorylation, and the sequence of Block was as follows:
SARS(ORF)-B：GCACGCGCGGGCAAGTCAATG-PO 4
SARS(N)-B：TGTCTGATAATGGACCCCAATCG-PO 4 。
2. the nucleic acid extraction-free fluorescent RT-PCR kit for detecting novel coronavirus 2019-nCoV nucleic acid of claim 1, wherein:
the enzyme mixture 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.
3. The nucleic acid extraction-free fluorescent RT-PCR kit for detecting novel coronavirus 2019-nCoV nucleic acid of claim 1, wherein:
the negative control is sterilized normal saline.
4. The nucleic acid extraction-free fluorescent RT-PCR kit for detecting novel coronavirus 2019-nCoV nucleic acid of claim 1, wherein:
the positive control was 10 3 copy/uL of pseudovirus comprising RNA sequences of the novel coronavirus open reading frame 1a/b gene, nucleocapsid protein gene and internal standard β -globin gene in the novel coronavirus 2019-nCoV nucleic acid.
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