CN111808988A - COVID-19 nucleic acid releasing agent and nucleic acid detection kit - Google Patents
COVID-19 nucleic acid releasing agent and nucleic acid detection kit Download PDFInfo
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Abstract
The invention relates to a COVID-19 nucleic acid releasing agent and a nucleic acid detection kit, belonging to the technical field of gene detection. The COVID-19 nucleic acid releasing agent comprises: surfactant with the mass volume percentage concentration of 0.01-1% and buffer reagent with the pH value of 6.0-8.8; the nucleic acid releasing agent can crack potential COVID-19 viruses of a sample to be detected on the basis of not destroying RNA of COVID-19 by matching the surfactant and the buffer reagent, release nucleic acid into a solution system, and replace 2-hour nucleic acid extraction. Therefore, the whole detection process is shortened from 4 hours to within 1.5 hours, and the method has the advantages of high detection speed, high sensitivity, strong specificity and good reliability.
Description
Technical Field
The invention relates to a gene detection technology, in particular to a COVID-19 nucleic acid releasing agent and a nucleic acid detection kit.
Background
The novel coronavirus pneumonia is an acute infectious pneumonia, and the pathogen is a novel coronavirus which is not found in human before, namely novel coronavirus (COVID-19). The virus has strong infectivity and pathogenicity, forms spreading trend in the world, has ever-increasing infection rate and death rate, seriously threatens the life safety of people and causes economic loss due to the influence of the spreading of the virus on various industries. Therefore, diagnosis and detection of novel coronaviruses are helpful to inhibit the development of epidemic situations.
Coronavirus is an enveloped RNA virus widely present in human, mammalian and avian hosts and can cause respiratory, intestinal, liver and nervous system diseases. For the detection of novel coronaviruses, there are currently immunological-based antibody detection kits and second-generation sequencing detection reagents. Although the detection means based on immunology has lower cost, the antibody detection has the defects of long generation window of the patient antibody, easy omission and low detection sensitivity and accuracy; the second-generation sequencing can perform sequencing analysis on the whole genome of the new coronavirus, the result is accurate, and new mutation can be found, but the second-generation sequencing detection virus has the defects of high detection cost, low speed and the like, and can not quickly diagnose the new coronavirus.
The real-time fluorescent quantitative PCR method (RT-PCR) has the advantages of low immunological basic cost and incomparable advantages of other methodologies such as high sensitivity, high accuracy and time period, so that the RT-PCR method is one of the best choices for detecting and diagnosing the novel coronavirus.
At present, a plurality of kits for detecting the novel coronavirus by adopting a fluorescent quantitative PCR technology exist in the market, and the detection process generally comprises the following steps: extracting nucleic acid of a novel coronavirus sample, carrying out reverse transcription of RNA, carrying out PCR detection, issuing a negative and positive report and the like. Although the detection sensitivity and accuracy of the kits are good, the whole detection process is long, the purpose of rapid diagnosis cannot be achieved, and particularly, in the epidemic outbreak period, a large number of samples wait for detection, and a solution with shorter detection time is needed.
Disclosure of Invention
In view of the above, it is necessary to provide a COVID-19 nucleic acid releasing agent, which can greatly shorten the detection time by applying the nucleic acid releasing agent to the RT-PCR method for detecting COVID-19.
A COVID-19 nucleic acid releasing agent comprising: surfactant with the mass volume percentage concentration of 0.01-1% and buffer reagent with the pH value of 6.0-8.8;
the surfactant is selected from: at least one of NP40, X-100, SDS, Tween 20;
the buffer reagent comprises Tris-HCl, KCl, NaCl, DTT and (NH)4)2SO4And spermidine.
The COVID-19 nucleic acid releasing agent is designed aiming at the structural characteristics of COVID-19, and the surfactant is used for achieving the effect of cracking cell membranes and virus protein shells and releasing virus nucleic acid; Tris-HCl in the buffer reagent ensures the pH stability of the reaction system and keeps the activity of the PCR amplification enzyme; DTT, spermidine, Na ions and the like can stabilize and enable virus RNA to gather, and can improve the activity of reverse transcriptase and amplification enzyme in the subsequent PCR reaction and improve the amplification efficiency; the proportion of each component of the surfactant and the buffer reagent is matched to enhance the PCR reaction effect, and finally, the potential COVID-19 virus of the sample to be detected can be cracked on the basis of not damaging the RNA of the COVID-19, and the nucleic acid is released into a solution system. The nucleic acid releasing agent can replace a nucleic acid extraction kit for use, and the whole virus detection process is shortened by more than 2 hours without nucleic acid extraction and purification.
In one embodiment, the surfactant has a mass volume percent concentration of 0.02-0.5%; the pH value of the buffer reagent is 6.5-8.0.
In one embodiment, the surfactant has a mass volume percentage concentration of 0.02-0.05%, and the buffer agent has a pH of 7.0-8.0.
In one embodiment, the concentration of KCl is 5 mM-100 mM, the concentration of NaCl is 1-20 mM, the concentration of DTT is 0.1-5 mM, and the concentration of (NH)4)2SO4The concentration of (A) is 2-20 mM, and the concentration of spermidine is 0.1-5%. The above mM means m mol/L.
In one embodiment, the concentration of KCl is 10 mM-50 mM, the concentration of NaCl is 5-10 mM, the concentration of DTT is 0.1-2 mM, and the concentration of (NH)4)2SO4The concentration of (A) is 5-10 mM, and the concentration of spermidine is 0.2-2%.
In one embodiment, the concentration of the KCl is 40 mM-50 mM, and the concentration of the NaCl is 5-8 mM; the concentration of the DTT is 1-1.5 mM; said (NH)4)2SO4The concentration of (A) is 5-8 mM; the concentration of spermidine is 1-1.5%.
The invention also discloses a COVID-19 nucleic acid detection kit which comprises the nucleic acid releasing agent and is used for RT-PCR detection.
When the nucleic acid releasing agent is used for RT-PCR detection, a nucleic acid extraction kit is not required to be adopted for extraction, the extracted nucleic acid is not required to be purified, and the whole virus detection process is shortened by more than 2 hours through optimization of steps.
In one embodiment, the nucleic acid detection kit further comprises:
a specific amplification primer pair and a specific fluorescent probe for amplifying the ORF1 region of the virus sequence;
a specific primer pair and a specific fluorescent probe for amplifying a virus N protein sequence; and
a specific amplification primer pair and a specific fluorescent probe of an endogenous reference gene RnaseP for monitoring and detecting the process.
In one embodiment, the nucleic acid detection kit further comprises RT-PCR reaction reagents, wherein the RT-PCR reaction reagents comprise: random primer containing 6 basic groups, dNTP, Tris-HCl, NaCl, KCl, DTT, reverse transcriptase, ATP, MgCl2And Taq enzyme.
The RT-PCR reaction reagent has 2 functions which are respectively as follows: 1) reverse transcribing the viral RNA into cDNA; 2) the specific amplification primer pair and the fluorescent probe are used for carrying out PCR detection on virus cDNA, so that the purpose of virus diagnosis is achieved.
The pH value of Tris-HCl is 7.0-9.0, and preferably, the pH value is 7.3-7.8; the concentration of the random primer is 100 nM-500 nM, preferably 150 nM-250 nM, more preferably 200 nM-250 nM; the concentration of dNTP is 100nM to 500nM, preferably 150nM to 250nM, more preferably 200nM to 250 nM; NaCl at a concentration of 1mM to 20mM, preferably at a concentration of 5mM to 15mM, more preferably at a concentration of 5mM to 10 mM; KCl is 10 mM-150 mM, preferably 50 mM-100 mM, more preferably 50 mM-60 mM; DTT concentration is 0.05-5 mM, preferably 0.1-2 mM, more preferably 0.1-1 mM; the concentration of the reverse transcriptase is 1-20U/muL, preferably 5-10U/muL, and more preferably 5-8U/muL; ATP concentration is 0.1-10 mM, preferably 1-5 mM, more preferably 1-2 mM; MgCl2A concentration of 1.0mM to 10mM, preferably a concentration of 1.5mM to 5.0mM, more preferably a concentration of 1.5mM to 3.0 mM; the concentration of the Taq enzyme is 0.01U/mu L-0.5U/mu L, and preferably, the concentration is 0.05-0.2U/mu L.
In one example, the specific amplification primer pair for amplifying the ORF1 region of the viral sequence is the following sequence:
ORF1a-F：AATGGTCATGTGTGGCGG TTCAC(SEQ NO.1)
ORF1a-R：CAGCTTGACAAATGTTAAAAACACTA(SEQ NO.2)
the specific fluorescent probe for amplifying the ORF1 region of the virus sequence has the following sequences:
ORF1a-P：CTCATCAGGAGATGC CACAACTGCT(SEQ NO.3)
the specific primer pair for amplifying the virus N protein sequence has the following sequences:
ORFN-F：CAGGCAGCAGTAGGGGAACTTCT(SEQ NO.4)
ORFN-R：GGCCTTTACCAGACATTTTGCTCTCA(SEQ NO.5)
the specific fluorescent probe for amplifying the virus N protein sequence has the following sequence:
ORFN-P：CTGCTCTTGCTTTGCTGCTGCTTGAC(SEQ NO.6)
the specific amplification primer pair of the endogenous reference gene RnaseP for monitoring the detection process has the following sequence:
RNaseP-F：CTGTGTGTCCACTCAGGCTTGT(SEQ NO.7)
RNaseP-R：ACAGATGGGTCTCAGGTGCAG(SEQ NO.8)
the specific fluorescent probe of the endogenous reference gene RnaseP for monitoring the detection process has the following sequence:
RNaseP-P：ACATTTCTCAGATCAGCATTTGCTGGCA(SEQ NO.9)。
the specific amplification primer pair and the fluorescent probe are designed aiming at a new coronavirus sequence conserved region and are designed and optimized in ORF1a1b and N protein regions, and the virus detection and diagnosis purposes of high detection reliability degree and good specificity are achieved.
In one embodiment, the specific amplification primer pair for amplifying the ORF1 region of the virus sequence, the specific amplification primer pair for amplifying the N protein sequence of the virus and the specific amplification primer pair for monitoring the endogenous reference gene RnaseP of the detection process are added with thio modification at the 3' end of the sequence. Through sulfo modification, the specificity of the primer can be enhanced, and the amplification efficiency is improved.
In one embodiment, the specific fluorescent detection probe is a Taqman hydrolysis probe, the 5 'end of which is labeled with a fluorescent substance, and the 3' end of which is labeled with a quenching substance. The fluorescent substance marked at the 5' end is one of FAM, VIC, HEX, CY3, CY5 and ROX; the quenching substance labeled at the 3' end is one of BHQ1, BHQ2 and BHQ 3.
Compared with the prior art, the invention has the following beneficial effects:
the COVID-19 nucleic acid releasing agent provided by the invention can crack potential COVID-19 viruses of a sample to be detected and release nucleic acid into a solution system on the basis of not destroying the RNA of COVID-19 through the matching of the surfactant and the buffer reagent, and can replace the 2-hour nucleic acid extraction.
According to the COVID-19 nucleic acid detection kit, the nucleic acid releasing agent is directly used for pretreatment, the pretreated product can be added into a reaction tube, the reverse transcription of cDNA and the fluorescence PCR detection react in a single tube, the separate operation of the reverse transcription of cDNA and the PCR detection is not needed, and the whole detection process can be shortened from 4 hours to within 1.5 hours by integrating the technical processes.
The COVID-19 nucleic acid detection kit has the advantages of high detection sensitivity, strong specificity and good reliability.
Drawings
FIG. 1 is a graph showing the results of ORF1a of 3 novel coronavirus samples in example 4;
FIG. 2 is a graph showing the results of N protein in 3 samples of the novel coronavirus in example 4;
FIG. 3 is a graph showing the results of ORF1a in two kits for the novel coronavirus sample 1 in example 5;
FIG. 4 is a graph showing the results of N proteins in two kits for the sample 1 of the novel coronavirus in example 5;
FIG. 5 is a graph showing the results of ORF1a in two kits for the novel coronavirus sample 2 in example 5;
FIG. 6 is a graph showing the results of N proteins in two kits for the sample 2 of the novel coronavirus in example 5;
FIG. 7 is a graph showing the results of ORF1a in two kits for the novel coronavirus sample 3 in example 5;
FIG. 8 is a graph showing the results of N proteins in two kits of the new coronavirus sample 3 in example 5;
FIG. 9 is a graph showing the results of specificity of ORF1a in the specificity test in example 6;
FIG. 10 is a graph showing the results of the specificity of the N protein in the specificity test in example 6;
FIG. 11 is a graph showing the results of ORF1a detection by the kit of the present invention in comparison of performance in example 7;
FIG. 12 is a graph showing the results of detection of N protein by the kit of the present invention in comparison of performances in example 7;
FIG. 13 is a graph showing the results of ORF1a detection by the crude extraction kit in comparison of performance in example 7;
FIG. 14 is a graph showing the results of N protein detection by the crude extraction kit in the performance comparison of example 7.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The mass-volume percentage concentration of the invention is m/v percentage concentration, namely 1% is 1g/100 mL.
Example 1
A COVID-19 nucleic acid releasing agent comprising: surfactant with mass volume percentage concentration of 0.02-0.05%, and buffer reagent with pH value of 7.0-8.0;
the surfactant is selected from: NP40, X-100, and Tween 20.
The buffer reagent comprises Tris-HCl, KCl, NaCl, DTT and (NH)4)2SO4And spermidine.
The concentration of the KCl is 40 mM-50 mM, and the concentration of the NaCl is 5-8 mM; the concentration of the DTT is 1-1.5 mM; said (NH)4)2SO4The concentration of (A) is 5-8 mM; the concentration of spermidine is 1-1.5%.
Example 2
A COVID-19 nucleic acid detection kit, comprising:
1) the nucleic acid releasing agent of example 1;
2) the specific primer pair and the specific fluorescent probe of the endogenous reference gene RnaseP for amplifying the ORF1 region and the N protein sequence of the virus sequence and monitoring the detection process have the working concentration of 10 mu M (10 mu mol/L), and are shown in the following table.
TABLE 1 specific primer pairs and specific fluorescent probe primer sequences
| primer/Probe name | Sequence (5 '-3') | Sequence numbering |
| ORF1a-F | AATGGTCATGTGTGGCGGTTCAC | SEQ NO.1 |
| ORF1a-R | CAGCTTGACAAATGTTAAAAACACTA | SEQ NO.2 |
| ORFN-F | CAGGCAGCAGTAGGGGAACTTCT | SEQ NO.4 |
| ORFN-R | GGCCTTTACCAGACATTTTGCTCTCA | SEQ NO.5 |
| RNaseP-F | CTGTGTGTCCACTCAGGCTTGT | SEQ NO.7 |
| RNaseP-R | ACAGATGGGTCTCAGGTGCAG | SEQ NO.8 |
| ORF1a-P | CTCATCAGGAGATGC CACAACTGCT | SEQ NO.3 |
| ORFN-P | CTGCTCTTGCTTTGCTGCTGCTTGAC | SEQ NO.6 |
| RNaseP-P | ACATTTCTCAGATCAGCATTTGCTGGCA | SEQ NO.9 |
3) RT-PCR reaction reagent: contains random primer with 6N basic groups, dNTP, Tris-HCl, NaCl, KCl, DTT, reverse transcriptase, ATP, MgCl2Taq enzyme, etc.
Wherein the pH value of Tris-HCl is 7.3-7.8; the concentration of the random primer is 200 nM-250 nM; the concentration of dNTP is 200 nM-250 nM; the NaCl concentration is 5 mM-10 mM; KCl concentration is 50 mM-60 mM; the concentration of DTT is 0.1-1 mM; the concentration of the reverse transcriptase is 5-8U/mu L; ATP concentration is 1 mM-2 mM; MgCl2The concentration is 1.5-3.0 mM; the concentration of the Taq enzyme is 0.05-0.2U/mu L.
Example 3
The kit of example 2 is used for detecting nucleic acid of COVID-19, and the method comprises the following steps:
1) viral nucleic acid release:
taking 10 μ L throat swab sample, adding 10 μ L nucleic acid releaser, mixing, and standing at room temperature for 10 min.
2) Preparing an RT-PCR system:
adding the sample (20 mu L) treated in the step 1) into an RT-PCR reaction system, wherein the RT-PCR reaction is 50 mu L, and the RT-PCR reaction system comprises 5 mu L of a primer/probe combination (each primer and probe in the table 1 of the example 3), 2 mu L of an enzyme solution (reverse transcriptase and Tag enzyme) and 23 mu L of a reaction buffer (the rest components in the RT-PCR reaction reagent of the example 2); and (4) subpackaging the RT-PCR reaction system into 96-well plates.
3) Putting the 96-well plate containing the RT-PCR reaction system prepared in the step 2) into a Roche 480 or ABI7500 instrument, setting a reaction program, and starting real-time detection.
4) And (4) analyzing results: and judging the result according to the Ct value of the amplification reaction result and the amplification curve.
Example 4
3 positive clinical pharyngeal swab specimens, validated as COVID-19, were tested as in example 3.
The experimental results are shown in FIGS. 1-2, in which 3S curves are the results of ORF1a sequence region and N protein sequence region of 3 positive samples, respectively, and the straight line is the negative control graph. FIG. 1 is a graph showing the results of ORF1a of 3 novel coronavirus samples; FIG. 2 is a graph showing the results of N protein in 3 new coronavirus samples, from which it can be seen that 3 positive new coronavirus samples were successfully detected and the negative control had no amplification curve.
Example 5
The performance of the kit of example 3 was compared to a conventional viral RNA extraction kit.
Firstly, a method is provided.
1. And (4) processing the sample.
Taking 3 positive new coronavirus samples, and averagely dividing the samples into 2 parts (namely two samples 1, two samples 2 and two samples 3 respectively), wherein each part is 200 mu L; wherein the first part is subjected to RNA extraction using a viral RNA extraction Kit (QIAamp MinEluteVirus Spin Kit (50) from Qiagen) in an elution volume of 50. mu.L; secondly, 10. mu.L of the sample was mixed with 10. mu.L of the nucleic acid releasing agent of example 1 by the method of example 4 without extraction, and left at room temperature for 10 minutes.
2. And (6) detecting.
Adding 2.5 mu L of virus RNA extracted from the first part into a reaction system of a conventional commercial coronavirus detection kit (2019-nCoV nucleic acid detection kit (multiplex fluorescence RT-PCR method) (100 persons/bag) of Nanjing Nodezaki Biotech Co., Ltd.), and detecting by referring to a method of an instruction; secondly, the test was carried out in the same manner as in example 4.
And II, obtaining a result.
The results of the tests are shown in FIGS. 3 to 8. Fig. 3 is a result graph of ORF1a of two kits of a new coronavirus sample 1 (curve No. 1 is a curve of the kit), fig. 4 is a result graph of N proteins of two kits of a new coronavirus sample 1 (curve No. 1 is a curve of the kit of the present invention), fig. 5 is a result graph of ORF1a of two kits of a new coronavirus sample 2 (curve No. 1 is a curve of the kit of the present invention), fig. 6 is a result graph of N proteins of two kits of a new coronavirus sample 2 (curve No. 1 is a curve of the kit of the present invention), fig. 7 is a result graph of ORF1a of two kits of a new coronavirus sample 3 (curve No. 1 is a curve of the kit of the present invention), and fig. 8 is a result graph of N proteins of two kits of a new coronavirus sample 3 (curve no.
As can be seen from the figure, the performance of the kit in example 2 is equivalent to that of the kit for extracting nucleic acid and then detecting RNA samples by using a conventional kit, and even an amplification curve appears earlier, so that the kit has better detection effect.
Example 6
The kit of example 2 was subjected to a detection specificity experiment.
Firstly, a method is provided.
Respectively selecting 1 new coronavirus sample, 1 influenza A virus sample, 1 respiratory syncytial virus and 1 EB virus, sucking 10 mu L of sample of each virus, respectively adding 10 mu L of nucleic acid releaser of the invention for mixing treatment, and standing at room temperature for 10 min; the assay was performed as in example 3.
And II, obtaining a result.
The results of the tests are shown in FIGS. 9-10. FIG. 9 is a graph showing the results of the specificity of ORF1a obtained by performing an experiment using the kit of example 2, wherein S-type curves are new coronavirus samples and the rest are other virus samples, and FIG. 10 is a graph showing the results of the specificity of N protein obtained by performing an experiment using the kit of example 2, wherein S-type curves are new coronavirus samples and the rest are other virus samples.
From the above results, it can be seen that the detection kit of example 2 has high specificity for detecting the COVID-19 virus, can distinguish different virus samples, and can be used in specific nucleic acid detection of COVID-19.
Example 7
The kit of example 2 was compared to the performance of a commercially available broad spectrum crude extract.
Firstly, a method is provided.
1. And (4) processing the sample.
Taking 3 positive new coronavirus samples, and averagely dividing the samples into 2 parts, wherein each part is 200 mu L; wherein the first part is obtained by using a broad-spectrum crude extraction reagent (TRIzol of invitrogen Co.)TMReagent (100ml)) and processed according to the instructions; secondly, according to the method of example 3, 10. mu.L of the sample was mixed with 10. mu.L of the nucleic acid releasing agent of example 1, and the mixture was left at room temperature for 10 minutes.
2. And (6) detecting.
Adding 20 mu L of a product treated by the first broad-spectrum crude reagent into the RT-PCR reaction system of the embodiment 3, and detecting according to the method of the embodiment 3; secondly, the test was carried out in the same manner as in example 3.
And II, obtaining a result.
The results of the tests are shown in FIGS. 11 to 14. FIG. 11 is a graph showing the results of ORF1a detection using the kit of example 2, FIG. 12 is a graph showing the results of N protein using the kit of example 2, FIG. 13 is a graph showing the results of ORF1a in a sample treated with the crude kit, and FIG. 14 is a graph showing the results of N gene in a sample treated with the crude kit.
As can be seen from the figure, the performance of the kit in example 2 is better than that of the kit which uses the conventional crude extraction kit and then performs detection, and the concrete expression is that the slope of the amplification curve of each sample is larger than that of the amplification curve of the broad-spectrum crude extraction reagent (the amplification curve is steeper) when the viral nucleic acid release reagent disclosed by the invention is matched with the RT-PCR reaction reagent disclosed by the invention, so that the amplification efficiency of the kit is higher; when the virus content of the sample is less, the kit still has a complete amplification curve, and the sample treated by the broad-spectrum crude extraction reagent has no amplification curve or the amplification curve is incomplete, so that the kit is higher in detection sensitivity, can effectively reduce the occurrence probability of false negative, and is more suitable for detecting new coronavirus.
Example 8
The kit of example 2 was used for bulk clinical specimen testing.
Firstly, a method is provided.
A batch of clinical samples were taken, 60 samples were counted, and each sample was divided into 2 parts on average. The first part adopts the kit of the embodiment 2 and carries out detection according to the method of the embodiment 3; secondly, a similar reagent kit is used for detection (a novel coronavirus (2019-nCoV) nucleic acid determination reagent kit (PCR-fluorescent probe method)) of Shengxiang biological science and technology Co., Ltd.
And II, obtaining a result.
Compared with other clinical evidences and cases finally confirmed by the detection method, the COVID-19 infection result obtained by detection according to the method has the advantages that the true positive rate of the kit is 100 percent, and the true negative rate is 100 percent; the true positive rate of the Shengxiang biological kit is 100 percent, and the true negative rate is 93.5 percent. The result shows that the kit has better detection sensitivity (100% of true positive rate) and better detection specificity and lower false negative rate (100% of true negative rate).
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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Claims (10)
1. A COVID-19 nucleic acid releasing agent comprising: surfactant with the mass volume percentage concentration of 0.01-1% and buffer reagent with the pH value of 6.0-8.8;
the surfactant is selected from: at least one of NP40, X-100, SDS, Tween 20;
the buffer reagent comprises Tris-HCl, KCl, NaCl, DTT and (NH)4)2SO4And spermidine.
2. The COVID-19 nucleic acid releasing agent of claim 1, wherein the surfactant is present at a concentration of 0.02 to 0.5% by weight; the pH value of the buffer reagent is 6.5-8.0.
3. The COVID-19 nucleic acid releasing agent of claim 1, wherein the surfactant is present at a concentration of 0.02 to 0.05% by weight, and the buffering agent has a pH of 7.0 to 8.0.
4. The COVID-19 nucleic acid releasing agent according to claim 1, wherein the concentration of KCl is 5mM to 100mM, the concentration of NaCl is 1mM to 20mM, the concentration of DTT is 0.1 mM to 5mM, and the concentration of (NH)4)2SO4The concentration of (A) is 2-20 mM, and the concentration of spermidine is 0.1-5%.
5. The COVID-19 nucleic acid releasing agent according to claim 1, wherein the concentration of KCl is 40mM to 50mM, and the concentration of NaCl is 5mM to 8 mM; the concentration of the DTT is 1-1.5 mM; said (NH)4)2SO4The concentration of (A) is 5-8 mM; the concentration of spermidine is 1-1.5%.
6. A COVID-19 nucleic acid detection kit comprising the nucleic acid releasing agent of any one of claims 1 to 5, wherein the nucleic acid detection kit is used for RT-PCR detection.
7. The COVID-19 nucleic acid detection kit of claim 6, comprising:
a specific amplification primer pair and a specific fluorescent probe for amplifying the ORF1 region of the virus sequence;
a specific primer pair and a specific fluorescent probe for amplifying a virus N protein sequence; and
a specific amplification primer pair and a specific fluorescent probe of an endogenous reference gene RnaseP for monitoring and detecting the process.
8. The COVID-19 nucleic acid detection kit of claim 7, further comprising RT-PCR reaction reagents comprising: random primer containing 6 basic groups, dNTP, Tris-HCl, NaCl, KCl, DTT, reverse transcriptase, ATP, MgCl2And Taq enzyme.
9. The COVID-19 nucleic acid detection kit of claim 7, wherein the specific amplification primer pair for amplifying the ORF1 region of the viral sequence is the following sequence:
ORF1a-F：AATGGTCATGTGTGGCGG TTCAC(SEQ NO.1)
ORF1a-R：CAGCTTGACAAATGTTAAAAACACTA(SEQ NO.2)
the specific fluorescent probe for amplifying the ORF1 region of the virus sequence has the following sequences:
ORF1a-P：CTCATCAGGAGATGC CACAACTGCT(SEQ NO.3)
the specific primer pair for amplifying the virus N protein sequence has the following sequences:
ORFN-F：CAGGCAGCAGTAGGGGAACTTCT(SEQ NO.4)
ORFN-R：GGCCTTTACCAGACATTTTGCTCTCA(SEQ NO.5)
the specific fluorescent probe for amplifying the virus N protein sequence has the following sequence:
ORFN-P：CTGCTCTTGCTTTGCTGCTGCTTGAC(SEQ NO.6)
the specific amplification primer pair of the endogenous reference gene RnaseP for monitoring the detection process has the following sequence:
RNaseP-F：CTGTGTGTCCACTCAGGCTTGT(SEQ NO.7)
RNaseP-R：ACAGATGGGTCTCAGGTGCAG(SEQ NO.8)
the specific fluorescent probe of the endogenous reference gene RnaseP for monitoring the detection process has the following sequence:
RNaseP-P：ACATTTCTCAGATCAGCATTTGCTGGCA(SEQ NO.9)。
10. the COVID-19 nucleic acid detection kit of claim 9, wherein the specific amplification primer pair for amplifying the ORF1 region of the virus sequence, the specific primer pair for amplifying the N protein sequence of the virus, and the specific amplification primer pair for monitoring the endogenous reference gene RnaseP of the detection process are all added with thio modification at the 3' end of the sequence.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112266910A (en) * | 2020-12-04 | 2021-01-26 | 南京求臻基因科技有限公司 | Nucleic acid releasing agent and nucleic acid releasing method thereof |
| US11214843B2 (en) | 2020-02-18 | 2022-01-04 | Life Technologies Corporation | Compositions, kits and methods for detection of viral sequences |
| CN114908149A (en) * | 2022-05-31 | 2022-08-16 | 因顿健康科技(苏州)有限公司 | Rapid gene detection method without nucleic acid extraction |
| CN115074356A (en) * | 2022-08-18 | 2022-09-20 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | New coronavirus nucleic acid extraction promoter and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160152656A1 (en) * | 2014-11-28 | 2016-06-02 | Kaohsiung Medical University | Methods of Using Phosphoramidation Reaction for Conjugation-Labeling Nucleic Acids |
| CN108642145A (en) * | 2018-05-18 | 2018-10-12 | 上海默礼生物医药科技有限公司 | A kind of reagent, amplification system and its amplification method and application for the real-time constant-temperature amplification of nucleic acid |
| CN111088408A (en) * | 2020-03-20 | 2020-05-01 | 广州凯普医药科技有限公司 | Detection kit for new coronavirus, influenza A and influenza B and respiratory syncytial virus |
| CN111172327A (en) * | 2020-03-05 | 2020-05-19 | 杭州丹威生物科技有限公司 | Method and kit for detecting novel coronavirus nucleic acid without taking hands |
-
2020
- 2020-06-09 CN CN202010518553.1A patent/CN111808988A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160152656A1 (en) * | 2014-11-28 | 2016-06-02 | Kaohsiung Medical University | Methods of Using Phosphoramidation Reaction for Conjugation-Labeling Nucleic Acids |
| CN108642145A (en) * | 2018-05-18 | 2018-10-12 | 上海默礼生物医药科技有限公司 | A kind of reagent, amplification system and its amplification method and application for the real-time constant-temperature amplification of nucleic acid |
| CN111172327A (en) * | 2020-03-05 | 2020-05-19 | 杭州丹威生物科技有限公司 | Method and kit for detecting novel coronavirus nucleic acid without taking hands |
| CN111088408A (en) * | 2020-03-20 | 2020-05-01 | 广州凯普医药科技有限公司 | Detection kit for new coronavirus, influenza A and influenza B and respiratory syncytial virus |
Non-Patent Citations (1)
| Title |
|---|
| 尹正君等: "《临床分子生物学检验技术》", 华中科技大学出版社, pages: 44 - 57 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11214843B2 (en) | 2020-02-18 | 2022-01-04 | Life Technologies Corporation | Compositions, kits and methods for detection of viral sequences |
| CN112266910A (en) * | 2020-12-04 | 2021-01-26 | 南京求臻基因科技有限公司 | Nucleic acid releasing agent and nucleic acid releasing method thereof |
| CN114908149A (en) * | 2022-05-31 | 2022-08-16 | 因顿健康科技(苏州)有限公司 | Rapid gene detection method without nucleic acid extraction |
| CN115074356A (en) * | 2022-08-18 | 2022-09-20 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | New coronavirus nucleic acid extraction promoter and application thereof |
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