CN111074008A - COVID-19 novel coronavirus nucleic acid detection method capable of improving accuracy - Google Patents
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Abstract
The invention relates to a COVID-19 novel coronavirus nucleic acid detection method capable of improving accuracy, which comprises the steps of mixing a reverse reaction system to carry out reverse reaction, and carrying out reverse transcription on polyA RNA into cDNA with poly (T) under the help of reverse transcriptase and oligo (dT); simultaneously carrying out RPA reaction on the reversed cDNA for a period of time for further amplification; mixing the amplified product with a QPCR system, performing chain reaction on a QPCR instrument, and respectively sequentially performing chain circulation reaction of denaturation, re-annealing and final extension; and collecting and detecting fluorescence signal intensity by detection equipment to perform qualitative or semi-quantitative analysis and determination on the target fragment, and finally judging the virus detection result of the patient according to the obtained different fluorescence signal data. The technical scheme can provide the COVID-19 novel coronavirus nucleic acid detection method which has strong specificity, accurate result and low false negative probability, can monitor each stage from the early stage to the later stage of infection of a patient and can improve the accuracy.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a novel COVID-19 coronavirus nucleic acid detection method capable of improving accuracy.
Background
The Novel coronavirus pneumonia ("Novel coronavirus pneumoconia", abbreviated as "NCP") is an acute respiratory infectious disease caused by 2019 infection of the Novel coronavirus (COVID-19). Approximately half of patients develop dyspnea after one week, and severe patients rapidly progress to acute respiratory distress syndrome, septic shock, refractory metabolic acidosis, and procoagulant dysfunction. The new coronavirus transmission routes including direct transmission, aerosol transmission and contact transmission have strong infectivity and long latency, so that great challenge is brought to epidemic prevention work.
The NCP is a single-stranded RNA virus, does not have a ribosome system required by RNA virus translation, and only after entering a host cell, protein translation is carried out by utilizing the RNA translation system of the host and taking virus genome RNA as a translation template, wherein the NCP comprises an RNA polymerase with multiple RNA replicase functional domains, and further the RNA polymerase is used for completing the transcription synthesis of minus-strand subgenomic RNA and the synthesis of mRNA of various structural proteins, so that the replication of the virus genome RNA is carried out, and then the RNA polymerase is assembled with the virus structural proteins and released to infect other cells. It follows that the invasion of viral RNA is the first step in viral infection. The kit detects the virus RNA of NCP to realize detection and tracking of NCP infection, and can evaluate whether a prognosis patient is recovered.
The means for the prior definite diagnosis of the new coronary pneumonia is to perform nucleic acid detection on viral genes by a fluorescent quantitative PCR method after viral RNA is inverted, and the current clinical practical use condition shows that the existing nucleic acid detection has more false negatives and brings great hidden danger to epidemic situation prevention and control. In addition, a colloidal gold detection method is adopted for the novel coronavirus (COVID-19), and the IgM and IgG antibodies are immune defense proteins generated after a human body is infected with the novel coronavirus, the IgG is an antibody which appears 14 days after infection and continuously exists after the generation and can be used as an index of previous infection, and the continuous existence of the IgG detected by the method after the infection can not accurately effectively evaluate the infection condition of a prognosis patient and accurately detect the health stage.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, including the problems that the existing fluorescence quantitative PCR method has more false negatives and the colloidal gold method can not accurately detect the infection condition of a prognosis patient to evaluate, and provides the COVID-19 novel coronavirus nucleic acid detection method which has strong specificity, accurate result and low false negative probability, can monitor each stage from the early stage to the later stage of the infection of the patient and can improve the accuracy.
In order to facilitate understanding of the technical terms involved in the technical solution, the following are organized:
COVID-19 is named for the new coronavirus model.
SSB refers to Single-Stranded Binding Protein (SSB) that plays an important role in DNA replication, recombination, and repair.
cDNA refers to DNA having a base sequence complementary to an RNA strand.
QPCR is short for the English full-name Real-time Quantitative PCR detection System, and refers to a Real-time fluorescence Quantitative nucleic acid amplification detection System, also called a Real-time Quantitative gene amplification fluorescence detection System.
A primer refers to a molecule having a specific nucleotide sequence, which stimulates the synthesis of a macromolecule having the specific nucleotide sequence at the start of nucleotide polymerization, and which is covalently linked to a reactant, and is referred to as a primer. The primers are typically two oligonucleotide sequences synthesized by man, one complementary to one of the DNA template strands at one end of the target region and the other complementary to the other DNA template strand at the other end of the target region, and function as the initiation point for nucleotide polymerization, from which 3 ends the nucleic acid polymerase can begin synthesizing new nucleic acid strands.
Deoxyribonucleotides are composed of one molecule of a nitrogen-containing base, one molecule of deoxyribose, one molecule of phosphate, wherein the 5 'position of ribose has a phosphate end, the 3' position is-OH (hydroxyl) and is hydroxyl end, and DNA can be linked, that is, the hydroxyl end of the former deoxyribonucleotide is linked with the phosphate end of the latter deoxyribonucleotide.
The 3' end of mRNA has a poly A tail with a length of 20-300 adenylates. The tail is involved in the movement of mRNA from the nucleus to the cytoplasm and also in the half-life of the mRNA. Studies find that the length of polyA is positively correlated with the mRNA lifetime, that ' freshly synthesized mRNA has a longer lifetime, and that ' old ' mRNA has a shorter lifetime; the mRNA synthesized in the first step of transcription is immature, a cap of a phosphate group is added at the 5 'end, which is mainly related to recognition and initiation of later translation, and a tail of polyA (poly A) is added at the 3' end to prevent the synthesized mRNA from being degraded, so that the mRNA has very important significance on the stability of the mRNA, and the mature mRNA can be called by splicing introns and the like.
poly refers to poly adenine ribonucleotides.
Oligo (dT) is a poly-thymine, T-repeat oligonucleotide, i.e., a nucleotide chain consisting of only thymine, producing only the desired cDNA, and oligo (dT) specifically binds to the poly (A) tail of mRNA, thereby specifically separating mRNA from total RNA.
phi29 DNA polymerase is a mesophilic DNA polymerase cloned from Bacillus subtilis phage phi 29. Has 3 'to 5' exonuclease proofreading capability and has special multiple replacement and continuous synthesis characteristics.
Where C represents Cycle and t represents threshold, and specifically refers to the number of cycles that the fluorescence signal in each reaction tube has reached a set threshold.
The P-value is the degree of risk to be overcome by the statistical colloquial understanding of the original hypothesis, a smaller value indicates a smaller risk, and a P-value is the probability of the appearance of a more extreme result than the obtained sample observation when the original hypothesis is true.
In order to achieve the purpose, the invention adopts the following technical scheme.
A method for detecting COVID-19 novel coronavirus nucleic acid capable of improving accuracy comprises the following steps:
step S1: mixing a proper amount of nucleic acid extracting solution serving as a sample to be detected with a reverse reaction system to perform reverse reaction, wherein the reverse reaction system comprises RNA reverse transcriptase, recombinase capable of combining single-stranded nucleic acid, single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase, so that RNA with 3' terminal polyA is reversely transcribed into cDNA with poly (T) under the help of the reverse transcriptase and oligo (dT);
step S2: simultaneously carrying out RPA reaction on the reversed cDNA for a period of time to further amplify, searching a homologous sequence in double-stranded DNA by adopting a protein-DNA compound formed by combining recombinase and a primer, carrying out strand exchange reaction on the primer after the primer is positioned to the homologous sequence to form and start DNA synthesis, carrying out exponential amplification on a target region on the template, and combining a replaced DNA strand with single-stranded DNA binding protein to prevent further replacement;
step S3: performing thermal denaturation on the target product amplified in the step S2, wherein R is a fluorescent group, Q is a quenching group, when Q is close to R, the probe does not emit light, the double DNA chains are separated, and the primer is combined with the single DNA chains during annealing;
step S4: the DNA polymerase is then combined with the double strand consisting of the primer and the single-stranded RNA, and the DNA polymerase starts extension at an extension temperature, wherein the DNA polymerase has exonuclease activity and cuts off the fluorescent group and the probe nucleotide when the DNA polymerase extends to the fluorescent end of the probe, so that the fluorescent group is separated from the quenching group;
step S5: mixing the amplified product with a QPCR system, performing chain reaction on a QPCR instrument, and respectively sequentially performing chain circulation reaction of denaturation, re-annealing and final extension;
step S6: and collecting and detecting fluorescence signal intensity by detection equipment to perform qualitative or semi-quantitative analysis and determination on the target fragment, and finally judging the virus detection result of the patient according to the obtained different fluorescence signal data.
As a further improvement of the invention, the primer in step S2 is an oligonucleotide primer.
As a further improvement of the present invention, the double-stranded DNA in step S2 is double-stranded after the primer is combined with the template cDNA.
As a further improvement of the invention, the detection device in the step S6 is used for carrying out fluorescence PCR detection by adopting qPCRmix.
As a further improvement of the present invention, the duration of the RPA reaction of the reversed cDNA in step S2 is set to fifteen minutes.
As a further improvement of the present invention, the number of chain circulation reactions in step S5 is selected to be in the range of thirty-five to forty.
As a further improvement of the present invention, the strand displacement DNA polymerase employs Phi29 strand displacement DNA polymerase having high continuous synthesis and strand displacement ability and having 3'-5' exonuclease activity.
As a further improvement of the present invention, the grade of the template molecule amplification product in the step S2 is in the order of five to ten times.
As a further improvement of the present invention, the template molecule amplification of step S2 may also employ any one of loop-mediated isothermal amplification, nucleic acid sequence-dependent amplification, rolling circle amplification, single primer isothermal amplification, helicase-dependent DNA isothermal amplification technique and strand displacement amplification.
Due to the application of the technical scheme, the technical scheme of the invention has the following beneficial effects: the invention can obviously improve the sensitivity of COVID-19 nucleic acid detection, and products produced by the method can be used for the confirmation of patients and the evaluation and judgment of emotional conditions of patient prognosis, because the invention adopts the RPA method to carry out isothermal amplification, 5-10 orders of magnitude of signal amplification can be carried out on the basis of common reversion, thus greatly enlarging the sensitivity of nucleic acid detection, and meanwhile, the normal reaction time length is not added on the basis of increasing the sensitivity, and the reversion-RPA reaction can be used for fluorescence detection after reacting for 10-15 minutes at 37 ℃; the invention directly detects the RNA of the virus, can directly reflect the virus infection condition without the influence of immune related antibodies in a receptor, and is obviously superior to a colloidal gold method; in the isothermal amplification process, the reversed cDNA signals are amplified by 5-10 orders of magnitude, so that the sensitivity of virus nucleic acid detection is greatly improved, and the reverse transcription time is not prolonged in the process of increasing the isothermal amplification; according to the technical scheme, the product subjected to signal amplification provides more templates for qPCR detection, so that the detection of a fuzzy sample can be effectively reduced, and the detection accuracy and sensitivity can be effectively improved.
Drawings
FIG. 1 is a schematic diagram of the RNA reverse transcription process of the present invention.
FIG. 2 is a schematic diagram of the RPA reaction process of the present invention.
FIG. 3 is a diagram of the qPCR reaction process of the present invention.
FIG. 4 is a diagram showing the comparison of qPCR results of the present invention and the existing kits in the market for detecting the same ginseng and ginseng.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
A method for detecting COVID-19 novel coronavirus nucleic acid capable of improving accuracy comprises the following steps: step S1: mixing a proper amount of nucleic acid extracting solution serving as a sample to be detected with a reverse reaction system to perform reverse reaction, wherein the reverse reaction system comprises RNA reverse transcriptase, recombinase capable of combining single-stranded nucleic acid, single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase, so that RNA with 3' terminal polyA is reversely transcribed into cDNA with poly (T) under the help of the reverse transcriptase and oligo (dT); step S2: simultaneously carrying out RPA reaction on the reversed cDNA for a period of time to further amplify, searching a homologous sequence in double-stranded DNA by adopting a protein-DNA compound formed by combining recombinase and a primer, carrying out strand exchange reaction on the primer after the primer is positioned to the homologous sequence to form and start DNA synthesis, carrying out exponential amplification on a target region on the template, and combining a replaced DNA strand with single-stranded DNA binding protein to prevent further replacement; step S3: performing thermal denaturation on the target product amplified in the step S2, wherein R is a fluorescent group, Q is a quenching group, when Q is close to R, the probe does not emit light, the double DNA chains are separated, and the primer is combined with the single DNA chains during annealing; step S4: the DNA polymerase is then combined with the double strand consisting of the primer and the single-stranded RNA, and the DNA polymerase starts extension at an extension temperature, wherein the DNA polymerase has exonuclease activity and cuts off the fluorescent group and the probe nucleotide when the DNA polymerase extends to the fluorescent end of the probe, so that the fluorescent group is separated from the quenching group; step S5: mixing the amplified product with a QPCR system, performing chain reaction on a QPCR instrument, and respectively sequentially performing chain circulation reaction of denaturation, re-annealing and final extension; step S6: and collecting and detecting fluorescence signal intensity by detection equipment to perform qualitative or semi-quantitative analysis and determination on the target fragment, and finally judging the virus detection result of the patient according to the obtained different fluorescence signal data.
The primers in the step S2 adopt oligonucleotide primers; the double-stranded DNA in the step S2 is double-stranded after the primer is combined with the template cDNA; the detection device in the step S6 adopts qPCRmix to carry out fluorescence PCR detection; the duration of the RPA reaction of the reversed cDNA in the step S2 is set to be fifteen minutes; the selection range of the number of the chain circulation reactions in the step S5 is thirty-five to forty; the chain replacement DNA polymerase adopts Phi29 chain replacement DNA polymerase which has high continuous synthesis and chain replacement capability and 3'-5' exonuclease activity; the grade of the template molecule amplification product in the step S2 is in the order of five to ten times.
Referring now to the following test examples to further illustrate, the reverse and RPA amplification reactions were first performed on the biological product solutions in the following table, which specifically includes the following steps:
and (3) uniformly mixing the reaction system, reacting for 15 minutes at a constant temperature of 37 ℃, and then cooling on ice.
Then qPCR reaction and result detection are carried out: mixing 10 mu L of qPCR mixed solution, 1 mu L of primer, 1 mu L of cDNA and 8 mu L of water, and reacting for 5 min at the temperature of 95 ℃; then carrying out reaction for 5s at 95 ℃, finally carrying out reaction for 20s at 60 ℃ (fluorescence detection stage), carrying out chain type circulation reaction for forty times, and finally keeping at 72 ℃ for 5 min; then, the detection equipment collects the intensity of the detected fluorescence signal to perform qualitative or semi-quantitative analysis and determination on the target fragment, and finally, the virus detection result of the patient is judged according to the obtained different fluorescence signal data.
The analysis and judgment of the detection data result are carried out according to the following indexes that the virus is determined to be positive when the Ct value is less than or equal to 35; when the Ct value is no value or is more than 38, and the detection result of the positive standard reference substance is positive, the result is judged that the new coronavirus (2019-nCoV) nucleic acid is negative; if the Ct value of the sample to be detected is more than 35 and less than or equal to 38, the sample is detected again after nucleic acid is extracted again.
To illustrate the accuracy and stability of the detection of the invention, the detection effect of the invention is verified by a comparison mode, the qPCR result of the detection of the same ginseng and ginseng by using the kit of the invention and the existing kit in the market is shown in figure 4 for the comparison of the result.
As shown in FIG. 4, curve 1 shows the results of the commercially available nucleic acid kit for detecting a positive ginseng sample; curve 2 is the same result of the positive ginseng sample detected by the kit of the invention; curve 3 is the result of the ginseng sample detected by the nucleic acid kit purchased in the market (repeated three times); curve 4 shows the results of the same scrophularia sample detected by the kit of the present invention (repeated three times); the abscissa in FIG. 4 represents the Ct number detected, and the ordinate represents the fluorescence intensity detected.
The detection time has a great influence on patient confirmation, in the reaction system of the invention, one synthesis event is started by two opposite primers, the whole process is very fast to be carried out, and amplification products with detectable levels can be obtained within fifteen minutes generally.
Therefore, the time required by detection is compared and analyzed, and the same male ginseng and female ginseng samples are detected by utilizing the kit and the existing nucleic acid detection kit in the market; the kit is sequentially carried out by adopting the steps set by the technical scheme, the total time consumption of detection operation is about 1 hour, the total time consumption of the existing kit in the market is about 1 hour, and the two kits are approximately equivalent in operation time.
The traditional nucleic acid detection kit in the market and the detection statistics and calculation results of the invention are as follows:
it can be seen from the above graph and statistical results that for the negative ginseng, the two kits both cannot detect the signal and do not have the reading (the circle marks in curve 3 and curve 4 in the curve result figure 4 and the result statistical table), showing that both have good detection capability for the negative ginseng; for the positive ginseng sample, the ct value of the kit of the invention is smaller relative to the ct value of the nucleic acid kit purchased in the market (curve 1 and curve 2 and result statistical table in fig. 4); therefore, when the same fluorescence intensity is obtained, the kit can reach the fluorescence value in a shorter cycle, the existing kit in the market needs 7-8 cycles, the sensitivity of the kit is more than 180 times higher than that of the existing kit in the market, and the kit has obvious difference through statistics, wherein the P value of less than 0.001 is the obvious difference.
From the results, the operation time of the two kits has no obvious difference for the same sample detection, but the sensitivity of the kit is far higher than that of the existing kit in the market, so that the detection accuracy of the novel virus RNA can be greatly improved; meanwhile, the detection of uncertain samples and false negative samples caused by the common nucleic acid detection kit can be further reduced in clinical detection.
The above is only a specific application example of the present invention, and the protection scope of the present invention is not limited in any way. All the technical solutions formed by equivalent transformation or equivalent replacement fall within the protection scope of the present invention.
Claims (9)
1. A method for detecting COVID-19 novel coronavirus nucleic acid capable of improving accuracy is characterized by comprising the following steps:
step S1: mixing a proper amount of nucleic acid extracting solution serving as a sample to be detected with a reverse reaction system to perform reverse reaction, wherein the reverse reaction system comprises RNA reverse transcriptase, recombinase capable of combining single-stranded nucleic acid, single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase, so that RNA with 3' terminal polyA is reversely transcribed into cDNA with poly (T) under the help of the reverse transcriptase and oligo (dT);
step S2: simultaneously carrying out RPA reaction on the cDNA reversed in the step S1 for a period of time to further amplify, searching a homologous sequence in double-stranded DNA by adopting a protein-DNA compound formed by combining recombinase and a primer, carrying out strand exchange reaction on the primer after positioning the homologous sequence to form and start DNA synthesis, carrying out exponential amplification on a target region on a template, and combining a replaced DNA strand with a single-stranded DNA binding protein to prevent further replacement;
step S3: performing thermal denaturation on the DNA double strand of the target product amplified in the step S2, wherein R is a fluorescent group, Q is a quenching group, when Q is close to R, the probe does not emit light, and the primer is combined with the single strand DNA during annealing;
step S4: the DNA polymerase then forms double-strand combination with the primer and the single-stranded RNA, and starts extension by the DNA polymerase at an extension temperature, wherein the DNA polymerase has exonuclease activity and cuts off the fluorescent group and the probe nucleotide when the DNA polymerase extends to the fluorescent end of the probe, so that the fluorescent group is separated from the quenching group;
step S5: mixing the amplified product with a QPCR reaction system, performing chain reaction on a QPCR instrument, and respectively and sequentially performing chain circulation reaction of denaturation, re-annealing and final extension;
step S6: and collecting and detecting fluorescence signal intensity by detection equipment to perform qualitative or semi-quantitative analysis and determination on the target fragment, and finally judging the virus detection result of the patient according to the obtained different fluorescence signal data.
2. The method for detecting COVID-19 coronavirus nucleic acid according to claim 1, wherein the method comprises the following steps: the primer in step S2 is an oligonucleotide primer.
3. The method for detecting COVID-19 coronavirus nucleic acid according to claim 1, wherein the method comprises the following steps: the double-stranded DNA in step S2 is double-stranded after the primer is bound to the template cDNA.
4. The method for detecting COVID-19 coronavirus nucleic acid according to claim 1, wherein the method comprises the following steps: the detection device in the step S6 adopts qPCRmix to carry out fluorescence PCR detection.
5. The method for detecting COVID-19 coronavirus nucleic acid according to claim 1, wherein the method comprises the following steps: the duration of the RPA reaction of the reversed cDNA in the step S2 was set to fifteen minutes.
6. The method for detecting COVID-19 coronavirus nucleic acid according to claim 1, wherein the method comprises the following steps: the number of chain circulation reactions in step S5 is selected to be in the range of thirty-five to forty.
7. The method for detecting COVID-19 coronavirus nucleic acid according to claim 1, wherein the method comprises the following steps: the chain replacement DNA polymerase adopts Phi29 chain replacement DNA polymerase which has high continuous synthesis and chain replacement capability and 3'-5' exonuclease activity.
8. The method for detecting COVID-19 coronavirus nucleic acid according to claim 1, wherein the method comprises the following steps: the grade of the template molecule amplification product in the step S2 is in the order of five to ten times.
9. The method for detecting COVID-19 coronavirus nucleic acid according to claim 1, wherein the method comprises the following steps: the template molecule amplification of step S2 may further employ any one of loop-mediated isothermal amplification, nucleic acid sequence-dependent amplification, rolling circle amplification, single primer isothermal amplification, helicase-dependent DNA isothermal amplification technique, and strand displacement amplification.
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| CN114058613B (en) * | 2021-11-18 | 2023-10-27 | 广州血液中心(中国医学科学院输血研究所广州分所、广州器官移植配型中心) | Large-volume high-sensitivity nucleic acid extraction method |
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