CN114561491A - Novel detection kit for coronavirus SARS-CoV-2, primer composition and detection method - Google Patents
Novel detection kit for coronavirus SARS-CoV-2, primer composition and detection method Download PDFInfo
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Abstract
The invention discloses a novel detection kit for coronavirus SARS-CoV-2, a primer composition and a detection method. The detection kit of the novel coronavirus SARS-CoV-2 comprises a first primer group aiming at the Orflab gene of the novel coronavirus SARS-CoV-2 and a second primer group aiming at the N gene of the novel coronavirus SARS-CoV-2; the first primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB, wherein the nucleotide sequences of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB are sequentially shown as SEQ ID No. 1-SEQ ID No. 6; the second primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP and a loop primer LB, the nucleotide sequences of which are sequentially shown as SEQ ID NO. 7-SEQ ID NO. 11. The detection kit, the primer composition and the detection method for the novel coronavirus SARS-CoV-2 have the advantages of high detection accuracy, good sensitivity, convenient operation and short detection time.
Description
Technical Field
The invention belongs to the field of detection kits, and relates to a detection kit, a primer composition and a detection method for novel coronavirus SARS-CoV-2, in particular to a detection kit for detecting novel coronavirus SARS-CoV-2 based on an LAMP method.
Background
The novel coronavirus pneumonia (COVID-19) is an acute respiratory infectious disease mainly caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2 or 2019-nCoV) infection, the clinical manifestations mainly include fever, hypodynamia and dry cough, and the upper respiratory symptoms such as nasal obstruction and rhinorrhea are rare. Approximately half of patients develop dyspnea after one week, with severe cases progressing rapidly to acute respiratory distress syndrome, septic shock, refractory metabolic acidosis, and hemorrhagic coagulation dysfunction.
At present, whether the new coronavirus is infected or not is mainly judged by carrying out in-vitro nucleic acid detection on the new coronavirus. Although the currently widely applied detection method, namely the fluorescence RT-PCR method, is currently widely applied, the method has certain defects in detection accuracy and sensitivity, needs large-scale precise instruments and professional technicians to operate, virtually causes the phenomena of overlong detection time and high detection cost, enables the detection of the new coronavirus to be operated and completed only in professional genetic testing institutions or hospitals, cannot become a field instant detection means of the new coronavirus, and cannot effectively relieve the problem that the detection of the new coronavirus is short of supply and demand at present.
Disclosure of Invention
In view of the above technical problems, the present invention aims to provide a novel detection kit, primer composition and detection method for SARS-CoV-2 coronavirus, which has high detection accuracy, good sensitivity, convenient operation and short detection time.
According to the first aspect of the invention, a detection kit of a novel coronavirus SARS-CoV-2 comprises a first primer group aiming at Orflab gene of the novel coronavirus SARS-CoV-2 and a second primer group aiming at N gene of the novel coronavirus SARS-CoV-2; the first primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB, wherein the nucleotide sequences of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB are sequentially shown as SEQ ID No. 1-SEQ ID No. 6; the second primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP and a loop primer LB, wherein the nucleotide sequences of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP and the loop primer LB are sequentially shown as SEQ ID No. 7-SEQ ID No. 11.
Nucleic acid sequence of a primer directed against the Orflab gene of SARS-CoV-2:
F3：5’-TGTTTTAAGCGGACACAATC-3’，SEQ ID NO.1；
B3：5’-GAACAGTATCTACACAAACTCTT-3’，SEQ ID NO.2；
FIP：5’-AGCACTCTTAAGAAGTTGAATGTCTTGCTAAACACTGTCTTCATG-3’，SEQ ID NO.3；
BIP：5’-AATCAGCACGAAGTTCTACTTGCTGTATAGGGTCAGCACCAA-3’，SEQ ID NO.4；
LF：5’-TCACCTTTGTTAACATTTGGGCC-3’，SEQ ID NO.5；
LB：5’-ACCATTATTATCAGCTGGTATTT-3’，SEQ ID NO.6。
nucleic acid sequence of a primer for the N gene of SARS-CoV-2:
F3：5’-CGGCAGTCAAGCCTCTTC-3’，SEQ ID NO.7；
B3：5’-TTGCTCTCAAGCTGGTTCAA-3’，SEQ ID NO.8；
FIP：5’-TCCCCTACTGCTGCCTGGAGCGTTCCTCATCACGTAGTCG-3’，SEQ ID NO.9；
BIP：5’-TTCTCCTGCTAGAATGGCTGGCTCTGTCAAGCAGCAGCAAAG-3’，SEQ ID NO.10；
LB：5’-GCGGTGATGCTGCTCTTG-3’，SEQ ID NO.11。
in a preferred embodiment, each of the primers is encapsulated separately. Dilution and mixing were performed before use. More preferably, the molar ratio of the primers Orflab-F3, Orflab-B3, Orflab-FIP, Orflab-BIP, Orflab-LF and Orflab-LB of the first primer group is preferably 1: 8: 4, so that the kit has a better amplification effect on the Orflab gene, obtains the lowest detection limit on the Orflab gene and improves the accuracy of a detection result. The preferred molar ratio of the primers N-F3, N-B3, N-FIP, N-BIP and N-LF in the second primer group is 1: 8: 4, so that the method has a good amplification effect on the N gene, obtains the lowest detection limit on the N gene, and improves the accuracy of a detection result. When in use, a primer mixed solution is prepared, and specifically comprises the following steps: dissolving FIP, BIP, B3, F3, LF and LB into solution respectively, mixing according to the ratio of F3: B3: FIP: BIP: LF: LB being 1: 8: 4, and if no LF primer is available, complementing by ddH 20.
In a preferred embodiment, each primer of the first primer set constitutes a first primer mixture; and the primers of the second primer group form a second primer mixed solution.
More preferably, in the first primer mixture, the molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB of the first primer group is 1: 8: 4; in the second primer mixed solution, the molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP and the loop primer LB of the second primer group is 1: 8: 4.
Further, the detection kit also comprises one or more of the following substances in combination: dNTPs, MgCl 210 × Isotermo Buffer, enzyme, 20 × EvaGreen Dye, template and ddH 20。
Furthermore, the kit also comprises a positive quality control product, wherein the positive quality control product comprises an armored virus containing an Orflab gene target fragment and an armored virus containing an N gene target fragment.
Further, the template comprises an armored virus containing an Orflab gene target fragment and an armored virus containing an N gene target fragment.
In a preferred embodiment, the interpretation criteria of the test kit are: if the fluorescence curves aiming at the Orflab gene and the N gene both have S-shaped curves, the Orflab gene is judged to be positive; if the fluorescence curves aiming at the Orflab gene and the N gene have no S-shaped curve, judging the gene is negative; if one of the fluorescence curves of the Orflab gene and the N gene has an S-shaped curve and the other does not have the S-shaped curve, the detection is carried out again, and if the fluorescence curves of the Orflab gene and the N gene still have the S-shaped curve and the other does not have the S-shaped curve, the detection is judged to be positive.
According to a second aspect of the present invention, a primer composition of a novel coronavirus SARS-CoV-2, comprising a first primer set for the Orflab gene of said novel coronavirus SARS-CoV-2 and a second primer set for the N gene of said novel coronavirus SARS-CoV-2; the first primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB, wherein the nucleotide sequences of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB are sequentially shown as SEQ ID No. 1-SEQ ID No. 6; the second primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP and a loop primer LB, wherein the nucleotide sequences of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP and the loop primer LB are sequentially shown as SEQ ID No. 7-SEQ ID No. 11.
According to the third aspect of the present invention, a method for detecting SARS-CoV-2, which is a novel coronavirus for non-diagnostic therapeutic purposes, comprises mixing a sample with the detection kit or the primer composition as described above, and then performing a loop-mediated isothermal amplification reaction.
In a preferred embodiment, the amplification reaction is performed at 60-65 ℃ for 18-30 min. More preferably, the procedure of the amplification reaction is a reaction at 65 ℃ for 20 min.
In a preferred embodiment, the Orflab gene in the sample is detected using a first amplification system, wherein: the first amplification system comprises the first primer group, dNTPs and MgCl 210 × Isotermo Buffer, enzyme, 20 × EvaGreen Dye, template and ddH 20. More preferably, the template comprises an armored virus comprising a fragment of the Orflab gene target.
In a preferred embodiment, the second amplification system is used to detect the N gene in the sample, wherein: the second amplification system comprises the second primer group, dNTPs and MgCl 210 × Isotermo Buffer, enzyme, 20 × EvaGreen Dye, template and ddH 20. More preferably, the template comprises an armored virus containing N gene target fragments.
More preferably, in the first amplification system or the second amplification system, the concentration of the primer composition is 0.2-2 μ M, the concentration of dNTPs is 1.0-2.0mM, and MgCl is added2The concentration of (B) is 5 to 10 mM.
More preferably, the molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB of the first primer set is 1: 8: 4, and the molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP and the loop primer LB of the second primer set is 1: 8: 4.
Specifically, during detection, the samples are respectively added into a first amplification system and a second amplification system, and loop-mediated isothermal amplification is respectively carried out, so as to synchronously detect whether the samples contain the Orflab gene and the N gene.
Compared with the prior art, the invention has the following advantages by adopting the scheme:
the detection kit, the primer composition and the detection method of the novel coronavirus SARS-CoV-2 are used for detecting based on a loop-mediated isothermal amplification method, 4 specific primers are respectively designed aiming at 6 regions of Orflab gene and N gene of SARS-CoV-2 virus, 2 or 1 loop primer is added, amplification is carried out in a constant temperature environment at 60-65 ℃, the sensitivity is good (the lowest detection limit can reach 10copies/mL and 50copies/mL), the operation is simple, and the time consumption is short; but also can detect the polygene locus of SARS-CoV-2 virus, thus improving the accuracy of detection; and the method can be operated by ordinary personnel without specialization without depending on expensive professional equipment, and provides possibility for the on-site instant detection of SARS-CoV-2 virus.
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In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a graph of the results of a minimum detection limit experiment for the SARS-CoV-2 virus Orflab gene target of the kit of example 2;
FIG. 2 is a graph of the results of the minimum detection limit experiment for SARS-CoV-2 virus N gene target using the kit of example 2.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the invention may be more readily understood by those skilled in the art. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto.
Reagents and materials required for the examples: the primers were synthesized by general biosystems (Anhui) Ltd; dNTPs were purchased from Solebao Biotechnology Ltd; nucleic acid Dye 20 × EvaGreen Dye was purchased from biotium; the new coronavirus and pseudovirus of the N gene of coronafab were synthesized by Shanghai Karma Biotech, Inc.
Example 1: design and screening of primer compositions for respiratory syncytial virus
In this example, primers for detecting the new coronavirus SARS-CoV-2 based on RT-LAMP were designed and screened, specifically including RT-LAMP primer set for detecting Orflab gene of the new coronavirus SARS-CoV-2 and RT-LAMP primer set for detecting N gene of the new coronavirus SARS-CoV-2, and the specific procedures are described below.
The NCBI downloads other common coronaviruses through the SARS-CoV-2 genome downloaded in GISAID, and designs the RT-LAMP primer group according to the common coronaviruses. The method specifically comprises the following steps: the specific Orflab fragment and N fragment sequences were obtained by comparative analysis of the sequences of the viruses HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, BioEdit and SnapGene.
(1) An online design software Primer Explorer V5 is utilized to design RT-LAMP primers for the specific Orflab gene fragment and the N gene fragment, 4 sets of primers are designed for the Orflab gene fragment, and 5 sets of primers are designed for the N gene fragment.
(2) ddH for Orflab target fragment-containing armored virus 20 is diluted in gradient with 10copies6copies/mL、105copies/mL、104copies/mL、103copies/mL. ddH for armored virus containing N gene target fragment 20 is diluted in gradient with 10copies6copies/mL、105copies/mL、104copies/mL、103copies/mL。
(3) The amplification reaction system is that the amplification primer Mix is 2.2 mu L, the dNTPs is 1.0-2.0mM, the MgCl 2: 5-10 mM, 2.5 muL of 10 × Isotermo Buffer, 0.2 muL of enzyme Mix, 1 muL of 20 × EvaGreen Dye, 5 muL of template, and adding ddH20 to make up to 25 muL, wherein the reaction program of the amplification primers is to react for 20min at 65 ℃. The template comprises two templates, the template of the amplification reaction system aiming at the Orflab gene is the armored virus containing the Orflab gene target fragment, and the template of the amplification reaction system aiming at the Orflab gene is the armored virus containing the N gene target fragment.
The Orflab gene primer information is shown in table 1.
TABLE 1
See table 2 for N gene primer information.
After the amplification reaction is finished, whether the fluorescence curve is positive or not is judged according to whether the fluorescence curve is an S-shaped curve or not, the S-shaped curve indicates that the detection result is positive, and the absence of the amplification curve indicates that the detection result is negative. The amplification results are shown in Table 3.
TABLE 3 Orflab Gene and N Gene primer screening amplification results
As can be seen from Table 3, the primer with the best amplification effect for the Orflab gene is the primer set 2, and the primer with the best amplification effect for the N gene is the primer set 3.
Example 2: detection kit
The detection kit comprises: first primer set, second primer set, dNTPs, MgCl 210 × Isotermo Buffer, enzyme, 20 × EvaGreen Dye, ddH 20. The first primer set is the primer set 2 in table 1, and the second primer set is the primer set 3 in table 2, each primer being independently encapsulated. The kit also comprises templates used as positive quality control products, and comprises two armored viruses which are respectively an Orflab gene target fragment and an N gene target fragment and are synthesized by Shanghai Kamin Biotech Co.
Before use, an amplification reaction system is prepared, and the method specifically comprises the following steps: 2.2. mu.L of amplification primer Mix, 1.4mM of dNTPs and MgCl28mM, 10 × Isothermo Buffer 2.5 μ L, enzyme Mix 0.2 μ L, 20 × EvaGreen Dye 1 μ L, template 5 μ L, ddH20 to 25 μ L. Wherein, two amplification reaction systems need to be prepared; the amplification primer Mix in the first amplification reaction system is a mixed solution of primers F3, B3, FIP, BIP, LF and LB aiming at the Orflab gene, and the template is an armored virus containing an Orflab gene target fragment; the amplification primer Mix in the second amplification reaction system is a mixed solution of primers F3, B3, FIP, BIP, LF and LB aiming at the N gene, and the template is armored virus containing the N gene target fragment.
In the first primer set for detecting the Orflab gene of the new coronavirus SARS-CoV-2, the molar ratio of the primers Orflab-F3, Orflab-B3, Orflab-FIP, Orflab-BIP, Orflab-LF and Orflab-LB is preferably 1: 8: 4. In the second primer group for detecting the N gene of the new coronavirus SARS-CoV-2, the molar ratio of the primers N-F3, N-B3, N-FIP, N-BIP and N-LF is preferably 1: 8: 4. A primer mixture was prepared in accordance with the molar ratio of each primer. Preparing an amplification primer Mix, dissolving FIP, BIP, B3, F3, LF and LB into a solution of 100umol/L, mixing according to the proportion of F3: B3: FIP: BIP: LF: LB: 1: 8: 4, and complementing ddH20 if no LF primer exists.
The using method of the kit comprises the following specific steps:
(1) sample treatment: taking a sample (such as a throat swab or an oral swab sample), transferring the sample into a centrifuge tube containing a sample lysate, shaking and uniformly mixing, and detecting;
(2) adding a first amplification system (20 mu L) and the liquid (5 mu L) in the step (1) into a test tube 1, adding a second amplification system (20 mu L) and the liquid (5 mu L) in the step (1) into a test tube 2, and reacting for 20min at 65 ℃ in a nucleic acid detector;
(3) and (4) interpretation of results: and judging whether the sample contains the new coronavirus according to whether the fluorescence curve is an S-shaped curve, wherein the S-shaped curve indicates that the detected sample contains the new coronavirus. In the embodiment, if the two targets of the new coronavirus Orflab gene are consistent, judging according to a consistent result; if the two targets are positive and negative, the detection is repeated, and if the detection result is negative and positive, the judgment is positive.
Feasibility of the kit
1. Limit of detection (LOD) test
Orflab target fragment-containing armored virus and ddH (recombinant human chorionic gonadotropin) for armored virus containing N gene target fragment 20 is diluted in a gradient way, and the copy number is 10 in sequence5copies/mL、104copies/mL、103copies/mL, 100copies/mL, 50copies/mL, 10 copies/mL. The Orflab target template was detected using the kit of example 2 according to the method of use described in example 2, and the N gene target template was detected using the kit of example 2 according to the method of use described in example 2, to determine the minimum amount of positive nucleic acid detected by the detection method. As shown in FIG. 1, the nucleic acid minimum detection limit of Orflab gene target of the new coronavirus SARS-CoV-2 is 10 copies/mL; as shown in FIG. 2, the lowest detection limit of nucleic acid of N gene target of the novel coronavirus SARS-CoV-2 is 50 copies/mL.
2. Experiment of specificity
(1) Specific experimental pathogen sample extraction: 13 viruses including influenza A virus (H1N1), influenza B virus (Yamagata), respiratory syncytial virus (A), adenovirus (2), parainfluenza virus (1), middle east respiratory syndrome coronavirus (MERSR-CoV), human coronavirus HCoV-229E, human coronavirus HCoV-HK U1, human coronavirus HCoV-NL63, human coronavirus HCoV-0C43, influenza A virus (H3N2), influenza A virus (H5N1), and influenza A virus (H7N9) were purified from the nucleic acid RNA genome of the Solebao Biotech Ltd using an RNA extraction kit.
(2) Sample detection: the detection of the 13 viral nucleic acids was carried out using the kits of example 2, Orflab gene target and N gene target template as positive controls, ddH 20 served as a negative control.
(3) And (4) analyzing results: referring to Table 4, influenza A virus (H1N1), influenza B virus (Yamagata), respiratory syncytial virus (type A), adenovirus (type 2), parainfluenza virus (type 1), middle east respiratory syndrome coronavirus (MERSR-CoV), human coronavirus HCoV-229E, human coronavirus HCoV-HK U1, human coronavirus HCoV-NL63, human coronavirus HCoV-0C43, influenza A virus (H3N2), influenza A virus (H5N1), and influenza A virus (H7N9) were not detected, indicating that the kit has high specificity.
TABLE 4 results of the specificity test
"+" indicates positive detection, and "-" indicates negative detection.
As used in this specification and the appended claims, the terms "comprises" and "comprising" are intended to only encompass the explicitly identified steps and elements, which do not constitute an exclusive list, and that a method or apparatus may include other steps or elements.
The singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and are preferred embodiments, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention. All equivalent changes or modifications made according to the principles of the present invention should be covered within the protection scope of the present invention.
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Claims (11)
1. A detection kit of novel coronavirus SARS-CoV-2, which is characterized by comprising a first primer group aiming at Orflab gene of the novel coronavirus SARS-CoV-2 and a second primer group aiming at N gene of the novel coronavirus SARS-CoV-2; the first primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB, wherein the nucleotide sequences of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB are sequentially shown as SEQ ID No. 1-SEQ ID No. 6; the second primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP and a loop primer LB, wherein the nucleotide sequences of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP and the loop primer LB are sequentially shown as SEQ ID No. 7-SEQ ID No. 11.
2. The test kit according to claim 1, wherein each of the primers is individually packaged.
3. The detection kit according to claim 1, wherein each primer of the first primer set constitutes a first primer mixture; and the primers of the second primer group form a second primer mixed solution.
4. The detection kit according to claim 3, wherein in the first primer mixture, the molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB in the first primer group is 1: 8: 4; in the second primer mixed solution, the molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP and the loop primer LB of the second primer group is 1: 8: 4.
5. The test kit of claim 4, further comprising a combination of one or more of the following: dNTPs, MgCl210 × Isotermo Buffer, enzyme, 20 × EvaGreen Dye, template and ddH2O。
6. The detection kit according to claim 1, wherein the interpretation criteria of the detection kit are: if the fluorescence curves aiming at the Orflab gene and the N gene both have S-shaped curves, the Orflab gene is judged to be positive; if the fluorescence curves aiming at the Orflab gene and the N gene have no S-shaped curve, judging the gene is negative; if one of the fluorescence curves for the Orflab gene and the N gene has an "S" -shaped curve and the other does not have an "S" -shaped curve, the re-detection is performed, and if one still has an "S" -shaped curve and the other does not have an "S" -shaped curve, the determination is positive.
7. A primer composition of a novel coronavirus SARS-CoV-2, comprising a first primer set for Orflab gene of the novel coronavirus SARS-CoV-2 and a second primer set for N gene of the novel coronavirus SARS-CoV-2; the first primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB, wherein the nucleotide sequences of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB are sequentially shown as SEQ ID No. 1-SEQ ID No. 6; the second primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP and a loop primer LB, wherein the nucleotide sequences of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP and the loop primer LB are sequentially shown as SEQ ID No. 7-SEQ ID No. 11.
8. A method for detecting SARS-CoV-2, a novel coronavirus as a non-diagnostic therapeutic object, which comprises carrying out a loop-mediated isothermal amplification reaction after mixing the sample with the detection kit according to any one of claims 1 to 6 or the primer composition according to claim 7.
9. The detection method according to claim 8, wherein the amplification reaction is carried out at 60 to 65 ℃ for 10 to 30 min.
10. The method according to claim 8, wherein the Orflab gene in the sample is detected using a first amplification system and the N gene in the sample is detected using a second amplification system, wherein: the first amplification system comprises the first primer group, dNTPs and MgCl210 × Isotermo Buffer, enzyme, 20 × EvaGreen Dye, template and ddH2O; the second amplification system comprises the second primer group, dNTPs and MgCl210 × Isotermo Buffer, enzyme, 20 × EvaGreen Dye, template and ddH2O。
11. The method according to claim 10, wherein the concentration of the primer composition in the first amplification system or the second amplification system is 0.1 to 2.0. mu.M, the concentration of dNTPs is 1.0 to 2.0mM, and MgCl is added2The concentration of (A) is 5-10 mM; and/or the molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB of the first primer group is 1: 8: 4, and the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP and the loop primer LB of the second primer group are/isThe molar ratio of LB is 1: 8: 4.
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