Novel coronavirus loop-mediated isothermal amplification rapid detection kit and use method thereof
阅读说明：本技术 新型冠状病毒环介导等温扩增快速检测试剂盒及使用方法 (Novel coronavirus loop-mediated isothermal amplification rapid detection kit and use method thereof ) 是由 不公告发明人 于 2020-04-10 设计创作，主要内容包括：本发明公开了一种用于新型冠状病毒检测的逆转录实时荧光定量环介导等温扩增试剂盒及其使用方法。所述试剂盒,由新型冠状病毒核衣壳(N)基因环介导等温扩增引物组构成,试剂盒成分主要由新冠病毒N基因环介导等温扩增引物组溶液、环介导等温扩增反应预混液、AMV逆转录酶、新冠病毒N基因阳性质控品构成,可采用逆转录实时荧光检测方法,进行新冠病毒的快速检测。本方法在30分钟之内可以完成检测,特异性强,灵敏度高,为新型冠状病毒的防控及流行趋势调查分析提供了一种简便、快速的途径。(The invention discloses a reverse transcription real-time fluorescence quantitative loop-mediated isothermal amplification kit for detecting novel coronavirus and a using method thereof. The kit is composed of a novel coronavirus nucleocapsid (N) gene loop-mediated isothermal amplification primer group, and the kit mainly comprises a novel coronavirus N gene loop-mediated isothermal amplification primer group solution, a loop-mediated isothermal amplification reaction premix solution, AMV reverse transcriptase and a novel coronavirus N gene positive quality control product, and can be used for rapidly detecting the novel coronavirus by adopting a reverse transcription real-time fluorescence detection method. The method can complete detection within 30 minutes, has strong specificity and high sensitivity, and provides a simple, convenient and quick way for prevention and control of the novel coronavirus and investigation and analysis of epidemic trend.)
1. A set of loop-mediated isothermal amplification primer group for detecting novel coronavirus (new coronavirus for short) is composed of a loop-mediated isothermal amplification primer group of a new coronavirus nucleocapsid (N) gene, and is characterized in that the loop-mediated isothermal amplification primer group can be used for loop-mediated isothermal amplification reaction for detecting the new coronavirus;
the loop-mediated isothermal amplification primer group of the N gene of the novel coronavirus is characterized by consisting of a pair of outer primers (SEQ No.1 and SEQ No. 2), a pair of inner primers (SEQ No.3 and SEQ No. 4) and a pair of loop primers (SEQ No.5 and SEQ No. 6), wherein the primer sequences are as follows:
。
2. The loop-mediated isothermal amplification primer group for the N gene of the new coronavirus according to claim 1, which is used for a new coronavirus detection kit and mainly comprises a solution of the loop-mediated isothermal amplification primer group for the N gene of the new coronavirus, a premixed solution of loop-mediated isothermal amplification reaction, AMV reverse transcriptase and a positive quality control product for the N gene of the new coronavirus, and is characterized in that the rapid detection of the new coronavirus can be carried out by adopting a reverse transcription real-time fluorescence detection method;
the loop-mediated isothermal amplification primer group solution for the N gene of the new coronavirus is characterized by being prepared from a pair of inner primers, a pair of outer primers and a pair of loop primers according to a certain proportion, wherein the inner primers and the outer primers are prepared according to the proportion of 2:1, 4:1, 8:1 and 16:1, the preferred proportion is 8:1, the proportion of the loop primers to the inner primers is 1:2, and the outer primers, the inner primers and the loop primers respectively have the following final reaction concentrations: 0.2. mu.M, 1.6. mu.M, 0.8. mu.M;
the premixed solution for the loop-mediated isothermal amplification reaction is characterized by comprising Bst polymerase, dNTP, betaine and MgSO4And a buffer solution, including a substance capable of causing the amplification product to emit a fluorescent signalCommercial kit isothermmal MasterMix (OptiGene, uk) or equivalent other kit;
the positive quality control product of the new coronavirus N gene is characterized in that the positive quality control product is an RNA template obtained by in vitro transcription and purification of a plasmid constructed by an artificial synthetic segment of the N gene region covering the amplification of the primer;
the conventional reaction system of the loop-mediated isothermal amplification kit for the N gene of the new coronavirus comprises a loop-mediated isothermal amplification primer group solution 6 mu L, a loop-mediated isothermal amplification reaction premixed solution 13.8 mu L reverse transcriptase 0.2 mu L, a sample to be detected or a positive quality control product 5 mu L, and the total amount is 25 mu L.
3. The loop-mediated isothermal amplification assay kit for the N gene of neocoronavirus of claim 2, wherein detection of neocoronavirus can be performed using real-time fluorescence detection; a loop-mediated isothermal amplification fluorescence detection system is used for realizing synchronous isothermal amplification and detecting a fluorescence signal in a reaction tube in real time to obtain an isothermal amplification curve and a dissolution curve so as to judge a detection result; the optimal amplification temperature is 64 ℃ after the optimal amplification result is that the amplification temperature is in the range of 60-67 ℃; the amplification procedure of the loop-mediated isothermal amplification real-time fluorescence detection system for the N gene of the new coronavirus is as follows:64℃，30min，98℃-80℃，0.05℃/s，10 min。
4. the kit for detecting N gene of coronavirus of claim 2, wherein the time between amplification (Peak time) corresponding to the maximum fluorescence signal of each reaction in real-time fluorescence detection and the logarithm of the initial copy number of in vitro transcription RNA template is 102Copy/reaction-107With a linear relationship, R, in the copy/reaction context2>0.970, can be used for determining the copy number of the template of the sample to be detected according to the standard curve(ii) a The detection limit of the method for detecting the RNA copy number of the sample template to be detected is 102Copying/reacting.
5. The method of using the kit for detecting the N gene of a novel coronavirus according to claim 2, wherein the method for analyzing and determining the amplification result comprises:
1) when an amplification curve detected by the loop-mediated isothermal amplification fluorescence detection system appears on a platform, the fluorescence value of the amplification curve is 5 times higher than a background noise signal and the dissolution curve is a single sharp peak, and the determination result is a positive result, and the determination result is a non-specific cross interference signal, wherein the fluorescence value of the amplification curve is 5 times higher than the background noise signal and the dissolution curve is not a single sharp peak; judging the result as negative if the fluorescence value is less than or equal to 5 times of the background noise signal;
2) when the N gene loop-mediated isothermal amplification reaction tube of the new coronavirus in the sample to be detected is a negative result, the new coronavirus is not detected; if the test sample to be tested is a positive result, the test sample is infected by the new coronavirus; the method is characterized in that the amplification curve of the N gene is based on the reading at 30 min.
Technical Field
The invention relates to a set of primer group, a kit and a use method for rapid virus detection, in particular to a set of loop-mediated isothermal amplification primer group, a kit and a use method for detecting novel coronavirus (new coronavirus for short), belonging to the field of new coronavirus detection.
Background
The new coronaviruses belong to the family of coronaviridae taxonomically (Corona viridae) Genus coronavirus (Corona virus) The viral genome comprises two flanking non-coding regions (5 '-UTR and 3' -UTR), one coding multi-protein reading frame consisting of 12 open reading frames (orf1ab, Spike, orf3a, orf3b, E, M, orf6, orf7a, orf7b, orf8/orf8b, N and orf9b), 1 leader sequence, 9 transcriptional control sequencesThe genome of which is arranged in the order of 5 '-replicase (orf1/ab) -structural protein [ spike (S) envelope (E) -membrane (M) -nucleocapsid (N) 3', the encoded products of which are replicase (orf1/ab), spike glycoprotein (S), envelope (E), membrane (M), nucleocapsid (N) and other hypothetical proteins in that order, but lack the hemagglutinin lipase (HE) gene specific to the β type coronavirus A line.
However, because the fluorescent quantitative RT-PCR reaction has certain dependence on temperature, a precise temperature control system is required, the detection time is long, the experiment cost is high, the method is not suitable for on-site rapid detection, and in the face of the world wide large-scale popularization of the new coronavirus, a rapid detection method is urgently needed to be developed, a loop-mediated isothermal amplification (L AMP) method is a novel nucleic acid amplification technology invented by Notomi doctor in Japan in 2000, has the advantages of simplicity, rapidness, high sensitivity, strong specificity and the like, and through the development of twenty years, the method is continuously improved, particularly in the aspect of result judgment, various visual methods and real-time fluorescent detection methods are widely applied, the aerosol pollution caused by electrophoretic uncovering can be controlled from the source, and the method is particularly suitable for on-site rapid detection and bedside detection.
However, because the fluorescent quantitative RT-PCR reaction has certain dependence on temperature, a precise temperature control system is required, the detection time is long, the experiment cost is high, the method is not suitable for on-site rapid detection, and in the face of the world wide large-scale popularization of the new coronavirus, a rapid detection method is urgently needed to be developed, a loop-mediated isothermal amplification (L AMP) method is a novel nucleic acid amplification technology invented by Notomi doctor in Japan in 2000, has the advantages of simplicity, rapidness, high sensitivity, strong specificity and the like, and through the development of twenty years, the method is continuously improved, particularly in the aspect of result judgment, various visual methods and real-time fluorescent detection methods are widely applied, the aerosol pollution caused by electrophoretic uncovering can be controlled from the source, and the method is particularly suitable for on-site rapid detection and bedside detection.
Disclosure of Invention
The invention mainly aims to provide a set of loop-mediated isothermal amplification kit for rapidly detecting new coronavirus.
The new coronavirus loop-mediated isothermal amplification primer group is designed aiming at a specific sequence of a SARS-CoV-2 nucleocapsid (N) gene. The N gene primer group comprises a pair of outer primers, a pair of inner primers and a pair of loop primers, and is prepared according to a certain proportion, wherein the inner primers and the outer primers are prepared according to the proportion of 2:1, 4:1, 8:1 and 16:1, the preferable proportion is 8:1, the proportion of the loop primers and the inner primers is 1:2, and the outer primers, the inner primers and the loop primers at the final reaction concentration are respectively as follows: 0.2. mu.M, 1.6. mu.M, 0.8. mu.M.
The pre-mixed solution of the loop-mediated Isothermal amplification reaction can be a commercial kit Isothermmal Master Mix (OptiGene company, UK) or other equivalent kit which can make the amplification product emit a fluorescence signal and consists of Bst polymerase, dNTP, betaine and MgSO4And a buffer solution. The kit also simultaneously introduces a new coronavirus N gene positive quality control product, which is an RNA template obtained by in vitro transcription and purification of a plasmid constructed by an artificial synthetic segment of an N gene region covering the amplification of a primer group.
The total volume of the new coronavirus N gene loop-mediated isothermal amplification reaction system is 25 mu L, wherein the total volume comprises N gene loop-mediated isothermal amplification primer group solution 6 mu L, loop-mediated isothermal amplification reaction premix liquid 13.8 mu L reverse transcriptase 0.2 mu L, and a sample to be detected or a positive quality control product 5 mu L, the optimal amplification temperature is 64 ℃ after the optimal result that the amplification temperature is in the range of 60-67 ℃, and the amplification program of the loop-mediated isothermal amplification fluorescence detection system is as follows:64℃，30min，98℃-80℃，0.05℃/s，10 min。
the new coronavirus N gene detection kit can be used for real-time fluorescence detectionThe amplification time (Peak time) corresponding to the maximum value of each reaction fluorescence signal in the method and the logarithm value of the initial copy number of the in vitro transcription RNA template are 102Copy/reaction-107With a linear relationship, R, in the copy/reaction context2>0.970, the copy number of the sample template to be detected can be determined according to the standard curve; the detection limit of the method for detecting the RNA copy number of the sample template to be detected is 102Copying/reacting.
The method for analyzing and judging the amplification result comprises the following steps:
1) when an amplification curve detected by the loop-mediated isothermal amplification fluorescence detection system appears on a platform, the fluorescence value of the amplification curve is 5 times higher than a background noise signal and the dissolution curve is a single sharp peak, and the determination result is a positive result, and the determination result is a non-specific cross interference signal, wherein the fluorescence value of the amplification curve is 5 times higher than the background noise signal and the dissolution curve is not a single sharp peak; judging the result as negative if the fluorescence value is less than or equal to 5 times of the background noise signal;
2) when the N gene loop-mediated isothermal amplification reaction tube of the new coronavirus in the sample to be detected is a negative result, the new coronavirus is not detected; if the test sample to be tested is a positive result, the test sample is infected by the new coronavirus; the method is characterized in that the amplification curve of the N gene is based on the reading at 30 min.
The application method of the loop-mediated isothermal amplification kit for diagnosing the new coronavirus SARS-CoV-2 also comprises the quantitative relation analysis of the amplification time (Peak time) corresponding to the maximum fluorescence signal and the initial copy number of the template. The method comprises respectively preparing the concentrations of 107Copy number/reaction, 106Copy number/reaction, 105Copy number/reaction, 104Copy number/reaction, 103Copy number/reaction, 102Copy number/reacted N gene positive quality control material water solution series, using reverse transcription loop-mediated isothermal amplification real-time fluorescence detection system to respectively detect positive quality control material water solution series, using reaction Peak time as ordinate, using in vitro transcription RNA template initial copy number logarithm value (L og (Copies/reaction)) as abscissa, establishing relation curve, using said curve, and measuring unknown sample Peak time to obtain the target sampleInitial copy number of the target template.
Drawings
FIG. 1 purity and integrity of RNA positive quality control product obtained by in vitro transcription of N gene of new coronavirus
FIG. 2 optimization of reaction conditions-primer concentration
FIG. 3 optimization of reaction conditions-amplification temperature
FIG. 4 shows the result of the primer specificity test of N gene of new coronavirus by reverse transcription loop-mediated isothermal amplification real-time fluorescence detection method.
In the figure, the template of 1 is new coronavirus N gene in vitro transcription RNA, the template of 2 is MERS-CoV in vitro transcription RNA, the template of 3 is SARS-CoV in vitro transcription RNA, the template of 4 is influenza A virus A/Shanghai Putuo/1203/2013 (H1N1pdm09) RNA, the template of 5 is influenza A virus A/Hangzhou/1/2013 (H7N9) RNA, the template of 6 is influenza A virus A/Environment/Jiangxi/21/2011 (H9N 2) RNA, the template of 7 is B/Beijing Haihua/1386/2013 (Victoria) RNA, the template of 8 is influenza B virus B/Chongqing YunZhongo/1361/2013 (Yamagata) RNA (1-3 template is constructed by the laboratory, and the template of 4-8 is given by the Chinese disease prevention and control center).
FIG. 5 shows the result of the sensitivity test of the N gene of the new coronavirus by reverse transcription loop-mediated isothermal amplification real-time fluorescence detection method.
In the figure, 1 is the preparation concentration 108Copy number/reaction, 2 formulation concentration 107Copy number/reaction, 3 to make up concentration 106Copy number/reaction, 4 is the preparation concentration 105Copy number/reaction, 5 is the preparation concentration 104Copy number/reaction, 6 is the preparation concentration 103Copy number/reaction, 7 is the preparation concentration 102Copy number/reaction, 8 is the water solution series of positive quality control product of new coronavirus N gene with the prepared concentration of 10 copy number/reaction.
FIG. 6 is a graph showing the relationship between the amplification time (Peak time) corresponding to the maximum fluorescence signal and the log of the initial copy number of the positive control template (L og (Copies/action)).
Detailed description of the invention
The apparatus and reagents used in this example are specifically as follows:
the instrument comprises the following steps:
GenieII (OptiGene, UK), Hitachi U-3310 ultraviolet-visible spectrophotometer.
Reagent:
isotermal Master Mix (OptiGene, UK), AMV reverse transcriptase (Promega, USA), Standard RNA Synthesis (NEB, USA), SalI endonuclease (NEB, USA), and all primers and plasmids are synthesized by general biology.
1. Test materials and methods
1.1 test materials
The synthetic fragment of the N gene of the new coronavirus is artificially synthesized by referring to a published sequence (MN 908947.3) on Genbank, is connected to a pGEM-T easy vector, is transformed into a competent cell DH5 α, and is stored in a 30% glycerol solution.
1.2 primer design
The N genes of the new coronavirus are compared by using DNAMAN software, a specific fragment aiming at the N genes is selected as a target sequence, and a loop-mediated isothermal amplification primer group of the N genes is designed by using online software PrimeExplorer V5 (http:// PrimeExplorer. jp/e /). The N gene loop-mediated isothermal amplification primer group consists of a pair of outer primers (SEQ No.1 and SEQ No. 2), a pair of inner primers (SEQ No.3 and SEQ No. 4) and a pair of loop primers (SEQ No.5 and SEQ No. 6), wherein the primer sequences are as follows:
1.3 extraction and validation of recombinant plasmids
Adding 100u L glycerol bacterial solution into L B culture medium with 3m L added with ampicillin in advance, carrying out shaking culture at 37 ℃ and 250rpm for 8-10h, extracting plasmid according to the instruction of the plasmid miniprep kit, carrying out enzyme digestion and PCR identification, adding 30% glycerol into the rest bacterial solution, and storing at-80 ℃.
1.4 preparation of in vitro transcribed RNA positive quality control
As shown by the result of plasmid sequencing, the artificially synthesized fragment of the N gene is directed by the T7 promoter. After the plasmid is cut and purified, an RNA mixture is prepared by using a T7 in-vitro transcription kit Standard RNA Synthesis, the RNA concentration is measured by using a Hitachi U-3310 ultraviolet-visible spectrophotometer after purification, and the copy number is calculated.
1.5 optimization of the reaction conditions
1.5.1 optimization of primer concentration
The primer is prepared by a pair of inner primers, a pair of outer primers and a pair of loop primers according to a certain proportion, wherein the inner primers and the outer primers are prepared according to the proportion of 2:1, 4:1, 8:1 and 16:1, the proportion of the loop primers and the inner primers is 1:2, and the optimal primer concentration proportion is selected.
1.5.2 optimization of amplification temperature
Setting the amplification temperature in the range of 60-67 ℃ and selecting the optimal reaction temperature.
1.6 Loop-mediated isothermal amplification reaction
The total volume of the loop-mediated isothermal amplification reaction system is 25 mu L, the reaction conditions comprise new coronavirus N gene primer mixed liquor 6 mu L (final concentration of reaction, outer primer, inner primer and loop primer are respectively 0.2 mu M, 1.6 mu M and 0.8 mu M), loop-mediated isothermal amplification reaction premixed liquor 13.8 mu L reverse transcriptase 0.2 mu L, reaction sample 5 mu L, negative control test replaces reaction sample with DEPC water, after sample addition, the reaction tube is placed in a loop-mediated isothermal amplification fluorescence detection system, the amplification procedure is as follows:64℃，30min，the fluorescence signal in the reaction tube is monitored in real time by the system at the temperature of 98-80 ℃ and at the temperature of 0.05 ℃/s for 10min, and the test result is judged according to the obtained isothermal amplification curve and the dissolution curve.
1.7 specificity test of primer mixture
Respectively taking N gene in vitro transcription RNA of the new coronavirus, MERS-CoV in vitro transcription RNA, SARS-CoV in vitro transcription RNA, influenza A virus A/Shanghai Putuo/1203/2013 (H1N1pdm09) RNA, influenza A virus A/Zhongzhou/1/2013 (H7N9) RNA, influenza A virus A/environment/Jiangxi/21/2011 (H9N 2) RNA, B/Beijing Haizhu/1386/2013 (Victoria) RNA and influenza B virus B/Chongqing Yuzhong/1361/2013 (Yamagata) RNA as reaction templates, and carrying out real-time fluorescence system detection by using N gene loop-mediated isothermal amplification primer mixed liquor of the new coronavirus to check the specificity of the primer mixed liquor.
1.8 quantitative relationship analysis of amplification time (Peak time) corresponding to the maximum fluorescence signal and initial copy number of template RNA
Respectively carrying out 10-fold gradient dilution on the in vitro transcription RNA with the measured concentration, and carrying out quality control on the RNA with each concentration by a real-time fluorescence detection system (the final concentration gradient is 10)8Copy number/reaction, 107Copy number/reaction, 106Copy number/reaction, 105Copy number/reaction, 104Copy number/reaction, 103Copy number/reaction, 102Copy number/reaction, 10 copy number/reaction). For each reaction assay result, a linear analysis was performed between the log value of the initial copy number of template RNA and Peak time in the real-time fluorescence assay.
2. Test results
2.1 identification of purity and integrity of RNA-positive quality control product transcribed in vitro
The fragment is inserted into the artificial synthetic plasmid, and the N gene is a DNA sequence with the size of 372 bp. In the in vitro transcription process, a T7 promoter sequence and a restriction enzyme region sequence are additionally added for about 140bp, and the obtained gene size is about 512 bp. The RNA purity and integrity of the RNA fragment obtained after in vitro transcription and purification are identified by 2% agarose gel electrophoresis, and as shown in FIG. 1 (M is RNA Marker, the third N is the in vitro transcription RNA fragment of the N gene of the new coronavirus), a single target band of the N gene can be seen within the range of 400-600bp, and the size is within the expected range.
2.2 optimization of primer concentration
As shown in FIG. 2, the inner primers and the outer primers are prepared according to the ratio of 2:1, 4:1, 8:1 and 16:1 (multiple wells), the ratio of the loop primers to the inner primers is 1:2, the optimal primer concentration ratio is selected, the optimal ratio of the outer primers, the inner primers and the loop primers is 1:8:4, and the final reaction concentrations are respectively as follows: the optimum reaction conditions were shown as curves of 0.2. mu.M, 1.6. mu.M and 0.8. mu.M.
2.3 optimization of amplification temperature
The amplification temperature was set in the range of 60-67 ℃ and the optimal reaction temperature was selected, the optimal amplification temperature being 64 ℃ as indicated in FIG. 3.
2.4 real-time fluorescence detection method primer specificity test results
The in vitro transcription RNA of the N gene of the new coronavirus, the in vitro transcription RNA of MERS-CoV, the in vitro transcription RNA of SARS-CoV, the A/Shanghai Putuo/1203/2013 (H1N1pdm09) RNA of the influenza A virus, A/Zhongzhou/1/2013 (H7N9) RNA of the influenza A virus, A/environment/Jiangxi/21/2011 (H9N 2) RNA of the influenza A virus, B/Beijing Haizhu/1386/2013 (Victoria) RNA of the influenza A virus and B/Chongqing Zhongzhong/1361/2013 (Yamagata) RNA of the influenza B virus are taken as reaction templates respectively, and the test result of the loop-mediated isothermal amplification real-time fluorescence detection of the N gene of the new coronavirus is shown in figure 4.
2.5 amplification time (Peak time) corresponding to the maximum fluorescence signal and the quantitative analysis result of the initial copy number of the in-vitro amplified RNA template gene of the positive quality control product
Sequentially carrying out 10-fold gradient dilution (the final reaction concentration gradient is 10) on the in vitro transcription RNA positive quality control product of the novel coronavirus N gene with the determined concentration8Copy number/reaction, 107Copy number/reaction, 106Copy number/reaction, 105Copy number/reaction, 104Copy number/reaction, 103Copy number/reaction, 102Copy number/reaction, 10 copy number/reaction), the RNA quality control products of the above concentrations were detected by a real-time fluorescence detection system, and the results are shown in fig. 5.
FIG. 6 shows the results of quantitative relationship analysis (the abscissa shows the logarithmic value of the initial copy number of template RNA in each reaction, and the ordinate shows the Peak time value of each reaction) at 102Copy number-107CopyingThe linear equation of the N gene of the new coronavirus in the number range is y = -1.895x +21.67, R2=0.970, which suggests that the amplification time of the fluorescent signal of the N gene of the new coronavirus reaching the maximum value is accelerated as the template content of the reaction system increases, and the linear relationship is better. The detection method can carry out quantitative detection, and the detection limit is 102Copying/reacting.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
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