CN113862398A - CAMP primer group and kit for amplifying SARS-CoV-2 - Google Patents
CAMP primer group and kit for amplifying SARS-CoV-2 Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to a CAMP primer group and a kit for amplifying SARS-CoV-2. The primer group comprises one or two of the following two primer groups: a primer group for amplifying S region and N region fragments of SARS-CoV-2. The invention also provides a kit for detecting SARS-CoV-2 virus. The invention uses the simulation DNA as the control detection process, and no false positive phenomenon is generated. The primer provided by the invention has high sensitivity and specificity, the provided visual kit provides great convenience for field detection, and the prepared kit can realize rapid and accurate detection of SARS-CoV-2.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a CAMP primer group and a kit for amplifying 2019 novel coronavirus (SARS-CoV-2).
Background
SARS-CoV-2 is a new type coronavirus which is separated and confirmed, and the virus pathogen has strong spreading property and rapid spreading speed. In a clinical study of 41 cases in early epidemic situations, SARS-CoV-2 patients account for 31.7% of the severe patients entering ICU, the total death rate of the disease reaches 14.6%, and the disease poses great threat to public health safety. China now classifies the infectious diseases as legal infectious diseases B and infectious diseases A for management. The novel Coronavirus pneumonia (coronavir disease 19, COVID-19) caused by SARS-CoV-2 has become a malignant infectious disease threatening the life health of all human beings. According to real-time statistical data of the world health organization, when the time reaches 2021, 9 and 19 days in Beijing, more than 2.28 million patients with the novel coronavirus pneumonia are diagnosed in a global accumulated way, and more than 460 million patients with death are diagnosed in a accumulated way. New types of coronaviruses are seriously threatening the health of human life and may be present in humans for a long time. The early and rapid diagnosis of new coronavirus infection plays a crucial role in limiting virus transmission and curing patients.
By 11/5/2020, the national drug administration approves 24 new coronavirus nucleic acid detection reagents, 25 antibody detection reagents and 2 antigen detection reagents. In the nucleic acid detection reagent, only 3 constant temperature detection reagents are used, and most of the reagents are used as a detection method by a fluorescence PCR method. The fluorescence PCR nucleic acid detection method has the advantages of high reliability and the like, but has the limitation factors of high requirement on test equipment, complex operation flow, long detection time, dependence on instrument interpretation results and the like, so that the detection reagent has certain difficulty in popularization and application in areas with low economy and special environments such as airports, stations and the like. Compared with fluorescence PCR detection, the nucleic acid isothermal amplification technology has the advantages of high specificity and high sensitivity, is simple to operate, does not depend on high-end instruments, is short in detection time, can directly judge the characteristics of detection results through naked eyes, and is a method very suitable for rapid field detection application with relatively short instrument facilities. The nucleic acid constant temperature detection methods popularized in the current market comprise RPA, LAMP and the like, which are foreign patents, and the application and popularization in the national market are limited.
The Competitive and complementary pairing-based isothermal amplification of nucleic acids (CAMP) is a method developed by the institute of Process engineering of the Chinese academy of sciences to replace the amplification of Japanese LAMP nucleic acids (see EP 3476938). The method carries out amplification under isothermal condition, utilizes a group of specific primers with different numbers from 2 to 6 to carry out specific recognition with a target sequence, finishes reaction within about 30 minutes, can add visual dye into a reaction system, and judges the result by observing the color of a reagent by naked eyes after the reaction is finished. Compared with other gene detection methods (such as fluorescence PCR, PCR and the like), the CAMP reaction can be completed in a constant-temperature water bath kettle without expensive instruments and equipment; the reaction result can be judged by naked eyes, and the result is not required to be judged by a gel electrophoresis method with various operation steps and time consumption; the operation is simple, and the test can be smoothly completed by personnel without professional skills, and the method is suitable for basic medical institutions and local inspection and quarantine departments. In addition, CAMP can also greatly shorten the operation time and reduce the sample pollution chance, and is suitable for the rapid diagnosis of COVID-19.
Disclosure of Invention
The invention provides a primer group for amplifying SARS-CoV-2 CAMP, a kit and application thereof in order to solve the problems, the primer provided by the invention has high sensitivity and specificity, and the preparation of the primer into the kit can realize the rapid and accurate detection of SARS-CoV-2.
The CAMP primer group for amplifying SARS-CoV-2 of the invention comprises a primer group for amplifying the S region segment of SARS-CoV-2 and/or a primer group for amplifying the N region segment of SARS-CoV-2.
Preferably, the fragments targeted by the primer set include an S region fragment for SARS-CoV-2 as shown in SEQ ID NO.13 and an N region fragment for SARS-CoV-2 as shown in SEQ ID NO. 14. Wherein, the primer group for the S region fragment amplification of SARS-CoV-2 comprises three pairs of primers, which are respectively: S-F2: the nucleotide sequence is shown as SEQ ID NO.1, S-R2: the nucleotide sequence is shown as SEQ ID NO. 2; and, S-NF: the nucleotide sequence is shown as SEQ ID NO.3, S-NR: the nucleotide sequence is shown as SEQ ID NO. 4; S-LF: the nucleotide sequence is shown as SEQ ID NO.5, S-LR: the nucleotide sequence is shown as SEQ ID NO. 6.
The primer group for amplifying the N region segment of SARS-CoV-2 comprises three pairs of primers, which are respectively: N-F2: the nucleotide sequence is shown as SEQ ID NO.7, N-R2: the nucleotide sequence is shown as SEQ ID NO. 8; and, N-NF: the nucleotide sequence is shown as SEQ ID NO.9, N-NR: the nucleotide sequence is shown as SEQ ID NO. 10; N-LF: the nucleotide sequence is shown as SEQ ID NO. 11; N-LR: the nucleotide sequence is shown in SEQ ID NO. 12.
The CAMP primer group provided by the invention aims at the specific conserved region (target gene) of SARS-CoV-2, and consists of 6 primers respectively aimed by the CAMP primer group, wherein the CAMP primer group comprises a sequence inner primer (NF/NR) group, an outer primer (F2/R2) group and a loop primer (LF/LR). Wherein, NF/NR is respectively an upstream inner primer and a downstream inner primer, and consists of an F2 region and an F1c region, the F2 region is complementary with the F2c region at the 3 'end of the target gene, and the F1c region has the same sequence as the F1c region at the 5' end of the target gene. F2/R2 are upstream and downstream outer primers, respectively, consisting of the F2 region, and are complementary to the F2c region of the target gene. The specific nucleotide sequences are shown in table 1 below:
TABLE 1 nucleotide sequence
The kit for detecting SARS-CoV-2 of the invention comprises the above-mentioned CAMP primer set. Preferably, the CAMP primer group can be put into ultrapure water to prepare a working solution. The working solution of the primer group can be a mixed solution containing 1-20 mu M of F2 and R2 primers and 5-80 mu M of NF and NR primers.
The kit further comprises a CAMP reaction solution and a CAMP color development solution. Further, the CAMP reaction solution comprises: bst polymerase, AMV reverse transcriptase, CAMP reaction buffer solution and ultrapure water. Wherein the CAMP reaction buffer solution comprises Tris-HCl, KCl and (NH)4)2SO4、MgSO4And Triton X-100.
Specifically, the CAMP reaction liquid system comprises the following components:
CAMP reaction solution (25 mu L system, solvent is ultrapure water)
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
2～20mM MgSO4
0.1～0.5％Triton X-100
0.2-1M betaine
1～1.6mM dNTP
5 ~ 10U Bst DNA polymerase (NEW ENGLAND Biolabs)
5 to 10U AMV reverse transcriptase (NEW ENGLAND Biolabs)
The CAMP color developing solution is preferably one of SYBR green I, Eva green, Hydroxyl Naphthol Blue (HNB), chrome black T (EBT) and the like.
For detection, for example, 10 to 15. mu.L of a primer set working solution and 25. mu.L of a CAMP reaction solution may be used. The adding amount of the CAMP color development liquid can be 100-150 mu mol/L detection mixed liquid.
The invention also provides a SARS-CoV-2 virus detection method for non-disease diagnosis purpose, comprising the following steps:
1) mixing a nucleic acid sample, a working solution of the CAMP primer group, a CAMP reaction solution, ultrapure water and a CAMP sealing solution to prepare an amplification reaction solution;
2) taking the prepared amplification reaction liquid, firstly reacting at 35-45 ℃ for 20-80 min, preferably 37 ℃, then reacting at 60-65 ℃ for 20-80 min, preferably 63 ℃ for 60min, and judging whether the sample contains SARS-CoV-2 virus according to the color development result.
According to the detection method, in the working solution of the CAMP primer group, the concentrations of primers S-F2 and S-R2 are both 0.2-0.4 mu M, the concentrations of primers S-NF and S-NR are both 1-2 mu M, and the concentrations of primers S-LF and S-LR are both 1-2 mu M; the concentration of the primers N-F2 and N-R2 is 0.2-0.4 mu M, the concentration of the primers N-NF and N-NR is 1-2 mu M, and the concentration of the primers N-LF and N-LR is 1-2 mu M;
the invention also provides the application of the CAMP primer group in the preparation of products for detecting SARS-CoV-2 virus.
The invention also provides the application of the CAMP kit for detecting SARS-CoV-2, in particular the application in SARS-CoV-2 virus detection for non-disease diagnosis.
Competitive complementary paired isothermal amplification of nucleic acids (CAMP) is a novel isothermal amplification of nucleic acids granted by European patent (EP 3476938) just after 4 months of 2020. The method comprises the steps of carrying out amplification under the isothermal condition of 63-65 ℃, specifically identifying a target sequence by utilizing a group of specific primers with different numbers from 2 to 6, completing reaction within about 30 minutes, adding a visual dye into a reaction system, and observing the color of a reagent by naked eyes after the reaction is finished to judge the result. The application carries out specific primer design and double-gene detection aiming at S and N protein coding sequences of SARS-CoV-2, and can effectively avoid the occurrence of missed detection caused by gene mutation of virus. In the process of detecting and screening SARS-CoV-2 virus for personnel or environment, if only one gene is detected to be positive, the sample is shown to be possibly infected with the virus, so that further confirmation detection is required, the occurrence of false negative detection results can be effectively avoided, and a more efficient detection method is provided for diagnosis and epidemic prevention.
Based on CAMP technology, the invention designs 2 groups of primers aiming at S and N specific conserved regions of SARS-CoV-2. The primer group provided by the invention has high sensitivity and strong specificity, and the kit prepared by the primer group can quickly and accurately detect SARS-CoV-2 contained in a sample to be detected. In addition, the primer group provided by the invention has extremely high specificity, so that the time required by CAMP amplification is short, the detection time is further shortened, and the operation is simplified. The method or the kit provided by the invention can complete the rapid and accurate detection of SARS-CoV-2 without expensive instruments and complex operation, so the method or the kit is suitable for the rapid field detection of airports, customs, ports, communities and the like with low professional degree during the outbreak of COVID-19. The visual kit provided by the invention provides great convenience for field detection, and the prepared kit can realize rapid and accurate detection of SARS-CoV-2.
Drawings
FIG. 1 is a graph showing the change of fluorescence intensity with reaction time in example 1 of the present invention.
FIG. 2 is a graph showing the change of fluorescence intensity with reaction time in example 2 of the present invention.
FIG. 3 is a diagram showing gel electrophoresis in example 3 of the present invention.
Fig. 4 is a schematic diagram of a visual detection result in embodiment 4 of the present invention.
The invention obtains the special subsidies (special subsidies) of the prevention and control of the new coronary pneumonia epidemic situation of the scientific fund after doctor China, and the subsidy numbers are as follows: 2020T130116ZX ".
Detailed Description
The experimental procedures used in the following examples are conventional unless otherwise specified, and may be specifically carried out by the methods specified in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to kits and product instructions; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 validation of amplification reaction for SARS-CoV-2S Using Eva Green
Eva Green is similar to SYBR Green I, is a dye with Green excitation wavelength and combined with all double helix minor groove regions of dsDNA, and has far less inhibition on nucleic acid amplification reactions such as PCR and the like. In the free state, Eva Green emits weak fluorescence, but once bound to double-stranded DNA, fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of Eva Green is correlated with the amount of double-stranded DNA, and the amount of double-stranded DNA present in the nucleic acid amplification system can be detected from the fluorescence signal.
Reaction solution combination (25. mu.L)
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X Eva Green(Biotum)
Primer:
200nM S-F2/SEQ ID NO.l
200nM S-R2/SEQ ID NO.2
1600nM S-NF/SEQ ID NO.3
1600nM S-NR/SEQ ID NO.4
800nM S-LF/SEQ ID NO.5
800nM S-LR/SEQ ID NO.6
the target is SARS-CoV-2-S dsDNA/SEQ ID NO.13
A control group without target was also set.
The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 1. The fluorescence detection is applied to the target that real-time monitoring can be realized, and the result can be judged in advance through a real-time amplification curve.
Example 2 validation of amplification reaction for SARS-CoV-2N Using Eva Green
Eva Green is similar to SYBR Green I, is a dye with Green excitation wavelength and combined with all double helix minor groove regions of dsDNA, and has far less inhibition on nucleic acid amplification reactions such as PCR and the like. In the free state, Eva Green emits weak fluorescence, but once bound to double-stranded DNA, fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of Eva Green is correlated with the amount of double-stranded DNA, and the amount of double-stranded DNA present in the nucleic acid amplification system can be detected from the fluorescence signal.
Reaction solution combination (25. mu.L)
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X Eva Green(Biotum)
Primer:
200nM N-F2/SEQ ID NO.7
200nM N-R2/SEQ ID NO.8
1600nM N-NF/SEQ ID NO.9
1600nM N-NR/SEQ ID NO.10
800nM N-LF/SEQ ID NO.11
800nM N-LR/SEQ ID NO.12
the target is SARS-CoV-2-N dsDNA/SEQ ID NO.14
A control group without target was also set.
The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 2. The fluorescence detection is applied to the target that real-time monitoring can be realized, and the result can be judged in advance through a real-time amplification curve.
Example 3 gel electrophoresis detection of amplification products of two 2019 New coronavirus (SARS-CoV-2) Gene target fragments
The amplification products obtained in examples 1 and 2 were recovered and subjected to gel electrophoresis detection. The samples were electrophoresed for 1 hour on a 90mV 1% agarose gel (TAE lysis) prestained in GelRed (Biotum). The results are shown in FIG. 3, where the numbers of the markers in each lane correspond to the following samples:
1: CAMP amplification product of SARS-CoV-2-S.
2: CAMP amplification product of SARS-CoV-2-N.
Example 4 visual detection of two 2019 New coronavirus Gene target fragments
Hydroxy Naphthol Blue (HNB) is an indicator applied to visual detection in CAMP technology. The principle is as follows: when the nucleic acid amplification reaction in vitro occurs, a large amount of insoluble magnesium pyrophosphate is produced in the reaction system, resulting in a decrease in magnesium ions in the system. HNB can display blue or violet in the solution of the amplification system due to different magnesium ion concentrations, so that the existence of the amplification reaction can be monitored by observing the change of the color of the solution of the reaction system. When the CAMP does not generate amplification reaction, the concentration of magnesium ions in the system is high, and the reaction liquid is violet; when the CAMP is subjected to amplification reaction, the concentration of magnesium ions in the system is low, and the reaction liquid is blue. The results are shown in FIG. 4.
Two reaction solutions for 2019 new coronavirus gene detection were carried out by the following primer combinations and reagents.
Reaction solution combination (25. mu.L)
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
120μM HNB；
The common reagents used above for visual detection were the following primers and target sequences used for each DNA fragment:
1) SARS-CoV-2-S gene detection primer:
200nM S-F2/SEQ ID NO.l
200nM S-R2/SEQ ID NO.2
1600nM S-NF/SEQ ID NO.3
1600nM S-NR/SEQ ID NO.4
800nM S-LF/SEQ ID NO.5
800nM S-LR/SEQ ID NO.6
the target is SARS-CoV-2-S dsDNA/SEQ ID NO.13
2) SARS-CoV-2-N gene detection primer:
200nM N-F2/SEQ ID NO.7
200nM N-R2/SEQ ID NO.8
1600nM N-NF/SEQ ID NO.9
1600nM N-NR/SEQ ID NO.10
800nM N-LF/SEQ ID NO.11
800nM N-LR/SEQ ID NO.12
the target is SARS-CoV-2-N dsDNA/SEQ ID NO.14
And (5) judging a result: blue is positive and purple is negative. The results are shown in FIG. 4, where the reference numbers for each reaction tube correspond to the samples as follows:
1: positive result of SARS-CoV-2-S gene detection
2: the SARS-CoV-2-S gene detection has negative result of the no-target control group.
3: positive result of SARS-CoV-2-N gene detection.
4: the SARS-CoV-2-N gene detection has negative result of the no-target control group.
The method can be realized by upper and lower limit values and interval values of intervals of process parameters (such as temperature, time and the like), and embodiments are not listed.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> institute of Process engineering of Chinese academy of sciences
<120> a CAMP primer group and kit for amplifying SARS-CoV-2
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aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180
aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240
aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300
ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360
aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420
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tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720
ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780
ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840
gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900
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gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080
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aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500
aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560
ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620
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ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa caacaaggcc 240
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Claims (9)
1. A CAMP primer set for amplifying SARS-CoV-2, wherein the CAMP primer set comprises a primer set for amplification of a segment of region S of SARS-CoV-2 and/or a primer set for amplification of a segment of region N of SARS-CoV-2.
2. The CAMP primer set of claim 1, wherein the primer set for amplification of the S region segment of SARS-CoV-2 comprises three pairs of primers, each pair comprising: S-F2: the nucleotide sequence is shown as SEQ ID NO.1, S-R2: the nucleotide sequence is shown as SEQ ID NO. 2; and, S-NF: the nucleotide sequence is shown as SEQ ID NO.3, S-NR: the nucleotide sequence is shown as SEQ ID NO. 4; S-LF: the nucleotide sequence is shown as SEQ ID NO. 5; S-LR: the nucleotide sequence is shown as SEQ ID NO. 6;
the primer group for amplifying the N region segment of SARS-CoV-2 comprises three pairs of primers, which are respectively: N-F2: the nucleotide sequence is shown as SEQ ID NO.7, N-R2: the nucleotide sequence is shown as SEQ ID NO. 8; and, N-NF: the nucleotide sequence is shown as SEQ ID NO.9, N-NR: the nucleotide sequence is shown as SEQ ID NO. 10; N-LF: the nucleotide sequence is shown as SEQ ID NO. 11; N-LR: the nucleotide sequence is shown in SEQ ID NO. 12.
3. A kit for detecting SARS-CoV-2, comprising the CAMP primer set of claim 1 or 2.
4. The kit according to claim 3, wherein the kit further comprises a CAMP reaction solution and a CAMP color development solution.
5. The kit of claim 4, wherein the CAMP reaction solution comprises: bst polymerase, AMV reverse transcriptase, CAMP reaction buffer solution and ultrapure water.
6. A method for detecting SARS-CoV-2 virus for non-disease diagnostic purposes, comprising the steps of:
1) mixing a nucleic acid sample, working solution of the CAMP primer group according to claim 1 or 2, CAMP reaction solution, ultrapure water and CAMP sealing solution to prepare amplification reaction solution;
2) and taking the prepared amplification reaction liquid, firstly reacting at the constant temperature of 35-45 ℃ for 5-10 min, then reacting at the temperature of 60-65 ℃ for 20-80 min, and judging whether the sample contains SARS-CoV-2 virus or not according to the color development result.
7. The detection method according to claim 6, wherein in the working solution of the CAMP primer set,
the concentration of the primers S-F2 and S-R2 is 0.2-0.4 mu M, and the concentration of the primers S-NF and S-NR is 1-2 mu M; the concentration of the primers S-LF and S-LR is 0.5-1 μ M.
The concentration of the primers N-F2 and N-R2 is 0.2-0.4 mu M, and the concentration of the primers N-NF and N-NR is 1-2 mu M; the concentration of the primers N-LF and N-LR is 0.5-1 μ M.
8. Use of the CAMP primer set of claim 1 or 2 for the preparation of a product for the detection of SARS-CoV-2 virus.
9. Use of a kit according to any of claims 3 to 5 for the detection of SARS-CoV-2 virus for non-disease diagnostic purposes.
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