CN112195275A - Primer group, kit and method for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus - Google Patents
Primer group, kit and method for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus Download PDFInfo
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Abstract
The invention relates to a primer group, a kit and a method for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus, wherein the primer group comprises: the influenza A virus amplification primer, the influenza B virus amplification primer and the 2019-nCOV virus amplification primer group are sequentially shown as SEQ ID NO: 5-SEQ ID NO:8 in sequence, the influenza A virus amplification primer F3, B3, FIP and BIP sequences are sequentially shown as SEQ ID NO: 9-SEQ ID NO:12 in sequence, the influenza B virus amplification primer F3, B3, FIP and BIP sequences are sequentially shown as SEQ ID NO:12 in sequence, the 2019-nCOV virus amplification primer group consists of one or two groups of N gene amplification primers and ORF1ab gene amplification primers, the N gene primer F3, B3, LF, LB, FIP and BIP sequences are sequentially shown as SEQ ID NO: 13-SEQ ID NO:18 in sequence, and the ORF1ab gene primer F3, B3, FIP and BIP sequences are sequentially shown as SEQ ID NO: 19-SEQ ID NO:22 in sequence. The method is simple and convenient to operate and good in adaptability, and whether the influenza A virus, the influenza B virus and the 2019-nCOV virus exist in a sample can be identified within 1h through single detection.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a primer group, a kit and a method for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus.
Background
Influenza virus (Influenza virus) belongs to Orthomyxoviridae (Orthomyxoviridae), is called Influenza virus for short, and comprises human Influenza virus and animal Influenza virus, wherein the human Influenza virus is divided into three types of A (A), B (B) and C (C), and is a pathogen of Influenza. Among them, influenza a virus can naturally infect humans and various animals, often suddenly occurs, spreads rapidly, and causes a worldwide pandemic many times. Influenza b viruses only infect humans in nature, generally in sporadic, outbreak, or small circulation.
2019-nCOV belongs to the genus coronavirus in the family of coronavirus, influenza A, influenza B and the novel coronavirus 2019-nCOV are single-stranded RNA viruses, and RNA replication and large-scale amplification are directly carried out through self-encoded RNA dependent RNA polymerase RDRP after the viruses invade a human body. Viral pathogens stimulate the immune system, produce a factor storm, and cause damage to body organs. The virus can cause respiratory tract infection, and similar symptoms such as fever, cough and the like appear, and can cause large-area spread in people through air droplets and other ways. The three early symptoms are not easily distinguished. Therefore, in patients with the above symptoms, if three different pathogens can be identified by nucleic acid detection means, it is of great significance to the control of diseases and the guarantee of life health and safety of people.
The traditional detection methods for influenza viruses and human coronaviruses are more, but generally have some defects, wherein the detection time for serology detection and antigen detection is short, the requirement is low, the sensitivity is not high, meanwhile, false positive is easy to cause, the virus separation and electron microscope detection are long, the method is complex, large-scale instruments and professional laboratories are needed, the molecular biology detection is a detection method with higher sensitivity at present, the nucleic acid detection aiming at the three pathogens mainly comprises a fluorescence quantitative PCR method, a loop-mediated constant temperature amplification method, a recombinase-mediated constant temperature amplification method and the like, wherein the fluorescence quantitative PCR method is high in detection channel throughput, but needs to rely on a fluorescence quantitative PCR instrument, the professional laboratories and detection personnel, the cost is high, and the popularization is not facilitated. The loop-mediated isothermal amplification method can efficiently amplify nucleic acid at constant temperature, and gets rid of the limitation of thermal cycle.
In the prior art, an invention patent with application publication number CN111057798A discloses a LAMP primer combination and a kit for detecting a novel coronavirus, and also discloses a method for detecting a novel coronavirus by using a primer group and a loop-mediated isothermal amplification method, but influenza a, influenza b and a novel coronavirus 2019-nCoV can not be detected simultaneously.
Disclosure of Invention
The first purpose of the invention is to provide a primer group for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus, and the primer group can be used for combined detection of influenza A virus, influenza B virus and novel coronavirus, so that the virus infected by a patient can be conveniently distinguished.
The second purpose of the invention is to provide a kit for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus.
The third purpose of the invention is to provide a method for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus.
In order to achieve the above purpose, the invention adopts the technical scheme that:
the LAMP combined primer group for detecting the influenza A virus, the influenza B virus and the novel coronavirus comprises: the influenza A virus amplification primer for detecting influenza A virus, the influenza B virus amplification primer for detecting influenza B virus and the 2019-nCOV virus amplification primer group for detecting novel coronavirus are disclosed, wherein the sequences of the influenza A virus amplification primer F3, B3, FIP and BIP are shown as SEQ ID NO:5 to SEQ ID NO:8, F3, B3, FIP and BIP sequences of the influenza B virus amplification primer are shown as SEQ ID NO:9 to SEQ ID NO:12, the 2019-nCOV virus amplification primer group consists of one or two groups of N gene amplification primers and ORF1ab gene amplification primers, wherein F3, B3, LF, LB, FIP and BIP sequences of the N gene primers are shown as SEQ ID NO:13 to SEQ ID NO:18, F3, B3, FIP and BIP sequences of the ORF1ab gene primer are shown as SEQ ID NO:19 to SEQ ID NO:22, respectively.
The kit for LAMP combined detection of the influenza A virus, the influenza B virus and the novel coronavirus comprises the primer group for LAMP combined detection of the influenza A virus, the influenza B virus and the novel coronavirus.
Further, the adding amount ratio of F3, B3, FIP and BIP of the influenza A virus amplification primer is 0.5:0.5:4:4, the adding amount ratio of F3, B3, FIP and BIP of the influenza B virus amplification primer is 0.5:0.5:4:4, the adding amount ratio of F3, B3, LF, LB, FIP and BIP of the N gene primer is 0.5:0.5:1:1:4:4, and the adding amount ratio of F3, B3, FIP and BIP of the ORF1ab gene primer is 0.5:0.5:4: 4.
Further, the kit also comprises an internal reference gene and an internal reference gene amplification primer for detecting the internal reference gene.
Furthermore, the reference gene is a human CALR gene, and the sequence of the human CALR gene is shown in SEQ ID NO. 23; the sequences of F3, B3, LF, LB, FIP and BIP of the internal reference gene amplification primer are shown as SEQ ID NO. 24-SEQ ID NO. 29 in sequence.
Further, the air conditioner is provided with a fan,also comprises five groups of reaction systems respectively corresponding to the influenza A virus amplification primer, the influenza B virus amplification primer, the N gene amplification primer, the ORF1ab gene amplification primer and the internal reference gene amplification primer, wherein each group of reaction systems comprises corresponding buffer solution and MgSO4Betaine, dNTPS, dUTP, chromogenic agent, Bst DNA polymerase, UNG enzyme and reverse transcriptase. The buffer solution is Thermo buffer, and the color developing agent is SYBR GREEN I.
Further, each group of the reaction system has a volume of 23ul and comprises the following substances: 2.5ul of buffer solution, 0.75-1.5 ul of MgSO42-4 ul of Betaine, 2.5-3.75 ul of dNTPS, 1ul of dUTP, 0.5ul of color developing agent, 1ul of Bst DNA polymerase, 0.125ul of UNG enzyme and 0.1ul of reverse transcriptase, and further comprises the following primers: 0.5ul of F3, 0.5ul of B3, 0 or 1ul of LF, 0 or 1ul of LB, 4ul of FIP, 4ul of BIP.
The LAMP combined detection method for the influenza A virus, the influenza B virus and the novel coronavirus comprises the following steps:
1) extracting nucleic acid from the purified sample;
2) preparing an LAMP reaction system, and carrying out loop-mediated isothermal amplification reaction;
3) as a result, the threshold was set at 90 cycles, i.e., 45 minutes, and the number of positive cells below the threshold was positive and the number of negative cells above the threshold was negative.
Further, the amplification condition is 60-65 ℃, 30-60 min, and a signal is collected for 30-60 s.
The invention has the beneficial effects that:
the primer group for LAMP combined detection of the influenza A virus, the influenza B virus and the novel coronavirus can be used for LAMP combined detection of the influenza A virus, the influenza B virus and the novel coronavirus, and is convenient for distinguishing which virus a patient is infected with.
The kit for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus does not need expensive instruments when used alone, and can be used by a conventional real-time constant-temperature fluorescence PCR instrument.
The kit for LAMP combined detection of the influenza A virus, the influenza B virus and the novel coronavirus is simple and convenient to operate and good in adaptability, and can identify whether the influenza A virus, the influenza B virus and the 2019-nCOV virus exist in a sample within 1h by single detection.
According to the kit for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus, the setting of the internal reference gene can effectively monitor the sample collection/sample storage/sample nucleic acid extraction process, and false negative is avoided.
The kit for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus has strong specificity and does not have cross reaction with other common respiratory pathogens. The sensitivity is high, wherein the detection limit of influenza A virus is 1000copies/mL, the detection limit of influenza B virus is 1000copies/mL, and the detection limit of 2019-nCoV is 1000 copies/mL.
Drawings
FIG. 1 shows the result of specific verification of the loop-mediated amplification system for influenza A virus;
FIG. 2 shows the detection sensitivity of the loop-mediated amplification system for influenza A virus;
FIG. 3 shows the result of specific verification of the loop-mediated amplification system for influenza B virus;
FIG. 4 shows the detection sensitivity of the loop-mediated amplification system for influenza B virus;
FIG. 5 shows the result of the specificity verification of the 2019-nCOV viral loop-mediated amplification system (N gene);
FIG. 6 shows the detection sensitivity (N gene) of 2019-nCOV viral loop-mediated amplification system;
FIG. 7 shows the result of verifying the specificity of the 2019-nCOV viral loop-mediated amplification system (ORF1ab gene);
FIG. 8 shows the detection sensitivity (ORF1ab gene) of the 2019-nCOV viral loop-mediated amplification system;
FIG. 9 shows the result of verifying the specificity of the internal reference CALR gene isothermal amplification reaction system.
Detailed Description
The present invention will be further described with reference to the following examples and accompanying drawings.
Example 1
The primer group and the kit for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus of the embodiment have the following reaction systems:
detection of influenza A virus
Influenza a virus specific gene segment: the gene is derived from an M gene of NCBI influenza A virus, and the sequence of the M gene is shown as SEQ ID NO. 1.
Primers for isothermal amplification of influenza a virus nucleic acid are shown in table 1. The sequences of F3, B3, FIP and BIP of the primers for isothermal amplification of influenza A virus nucleic acid are shown as SEQ ID NO. 5-SEQ ID NO. 8 in sequence.
TABLE 1 primers for influenza A virus specific isothermal amplification
| Primer name | Primer sequences |
| F3 | tggaaatggggatccaaaca |
| B3 | gcacacaccaggccaaat |
| FIP | gccacctcttttgctccatggacatggacagagcggtcaaac |
| BIP | tcaactggtgcacttgccagcccacttcagtggtcacagtc |
The reaction system for isothermal amplification of influenza a virus nucleic acid is shown in table 2.
TABLE 2 isothermal amplification reaction System for influenza A Virus nucleic acids
And (3) amplification procedure: signals were collected once at 63 ℃ for 60min for 30 s.
Primer specificity: crossover experiments were performed and the detection of pathogens for crossover is shown in table 3.
TABLE 3 pathogen species for carrying out the Cross-reactivity
| Endemic human coronavirus HKU1 | Endemic human coronavirus NL63 |
| Endemic human coronavirus OC43 | Parainfluenza virus |
| Endemic human coronavirus 229E | Mycoplasma pneumoniae |
| SARS coronavirus | Chlamydia pneumoniae |
| MERS coronavirus | Streptococcus pneumoniae |
| Influence-A | Klebsiella pneumoniae |
| H7N9 | Legionella bacteria |
| Influence-B | Human leukocyte |
| Respiratory syncytial virus type A | Adenoviral vectors |
| Rhinovirus | Neisseria meningitidis |
| Adenovirus type 1 | Metapneumovirus |
| Enterovirus A | Bocavirus |
| Human metapneumovirus | Staphylococcus aureus |
| EB virus | Mycobacterium tuberculosis |
| Measles virus | Haemophilus influenzae |
| Human cytomegalovirus | Mumps virus |
| Rotavirus virus | Varicella-zoster virus |
| Norovirus | 2019-nCOV |
The cross reaction result is shown in figure 1, and as can be seen from figure 1, the detection specificity is strong, and the cross reaction with other pathogens of common respiratory tract is avoided.
Detection sensitivity: the constructed M gene plasmid of influenza A virus is diluted by 10 degrees in a gradient way8copies/ml、 107copies/ml、106copies/ml、105copies/ml、104copies/ml、103copies/ml、102copies/ml、101The primers/ml, 1 primers/ml and 0 primers/ml were used as templates for the detection sensitivity verification, and the sensitivity detection results are shown in FIG. 2. As can be seen from FIG. 2, the detection sensitivity was 103copies/ml。
Detection of influenza B virus
Influenza b virus specific gene fragments: the gene is derived from NEP gene of influenza B virus. The NEP gene sequence of the influenza B virus is shown as SEQ ID NO. 2.
The primers used for isothermal amplification of influenza B virus nucleic acid are shown in Table 4. The sequences of F3, B3, FIP and BIP of the primer for constant temperature amplification of the influenza B virus nucleic acid are shown as SEQ ID NO 9-SEQ ID NO 12 in sequence.
TABLE 4 primers for influenza B virus specific isothermal amplification
| Primer name | Primer sequences |
| F3 | ttacccttcaaccccagg |
| B3 | gttccgtttactaacactctc |
| FIP | gcatctttgttgttcatgtcccttagatgacatagaagaagaaccag |
| BIP | taaacactcagaaagaagggaagttagggacaatacattacgcata |
The reaction system for isothermal amplification of influenza a virus nucleic acid is shown in table 5.
TABLE 5 isothermal amplification reaction System for influenza B Virus nucleic acids
And (3) amplification procedure: signals were collected once at 63 ℃ for 60min for 30 s.
Primer specificity: cross-reaction assays were performed to detect the pathogens as shown in Table 3.
The cross reaction result is shown in fig. 3, and as can be seen from fig. 3, the specificity of detection is strong, and the detection has no cross reaction with other pathogens of common respiratory tract.
Detection sensitivity: the constructed NEP gene plasmid of the influenza B virus is diluted by 10 degrees in a gradient way8copies/ml、 107copies/ml、106copies/ml、105copies/ml、104copies/ml、103copies/ml、102copies/ml、101copies/ml、 1copies/ml、0The sensitivity of detection is verified by taking copies/ml as a template, and the detection result of the sensitivity is shown in FIG. 4. As can be seen from FIG. 4, the detection sensitivity was 103copies/ml。
Detection of 2019-nCOV virus:
and detecting the N gene and the ORF1ab gene in the 2019-nCOV virus specific gene.
2019-nCOV virus specific gene fragment: the gene is derived from a 2019-nCOV virus N gene, and the sequence of the 2019-nCOV virus N gene is shown as SEQ ID NO. 3.
The primers used for isothermal amplification of 2019-nCOV viral nucleic acid are shown in Table 6. The sequences of F3, B3, LF, LB, FIP and BIP of the primer for constant temperature amplification of 2019-nCOV virus nucleic acid are sequentially shown as SEQ ID NO. 13-SEQ ID NO. 18.
TABLE 62019 primers for isothermal amplification of nCOV Virus specificity
| Primer name | Primer sequences |
| F3 | gccaaaaggcttctacgca |
| B3 | ttgctctcaagctggttcaa |
| LF | aatagcagtccagatgaccaaattg |
| LB | gcggtgatgctgctcttg |
| FIP | tcccctactgctgcctggaggcagtcaagcctcttctcg |
| BIP | tctcctgctagaatggctggcatctgtcaagcagcagcaaag |
The reaction system for isothermal amplification of 2019-nCOV viral nucleic acid is shown in Table 7.
TABLE 72019 isothermal amplification reaction System for nCOV viral nucleic acids
And (3) amplification procedure: signals were collected once at 63 ℃ for 60min for 30 s.
Primer specificity: crossover experiments were performed and the detection of pathogens for crossover is shown in table 3.
The cross reaction result is shown in fig. 5, and as can be seen from fig. 5, the specificity of detection is strong, and the cross reaction with other pathogens of common respiratory tract is avoided.
Detection sensitivity: carrying out gradient dilution on the constructed N gene plasmid of the 2019-nCOV virus by 108copies/ml、 107copies/ml、106copies/ml、105copies/ml、104copies/ml、103copies/ml、102copies/ml、101The primers/ml, 1 primers/ml and 0 primers/ml were used as templates for the detection sensitivity verification, and the sensitivity detection results are shown in FIG. 6. As can be seen from FIG. 6, the detection sensitivity was 103copies/ml。
2019-nCOV virus specific gene fragment: the gene is derived from the 2019-nCOV virus ORF1ab gene, and the sequence of the 2019-nCOV virus ORF1ab gene is shown as SEQ ID NO. 4.
The primers used for isothermal amplification of 2019-nCOV viral nucleic acid are shown in Table 8. The sequences of F3, B3, FIP and BIP of the primer for the constant temperature amplification of 2019-nCOV virus nucleic acid are sequentially shown as SEQ ID NO. 19-SEQ ID NO. 22.
TABLE 82019 primers for isothermal amplification of nCOV Virus specificity
The reaction system for isothermal amplification of 2019-nCOV viral nucleic acid is shown in Table 9.
TABLE 92019 isothermal amplification reaction System for nCOV viral nucleic acids
And (3) amplification procedure: signals were collected once at 63 ℃ for 60min for 30 s.
Primer specificity: crossover experiments were performed and the detection of pathogens for crossover is shown in table 3.
The cross-reaction result is shown in FIG. 7. As can be seen from FIG. 7, the specificity of detection is strong, and the cross-reaction with other pathogens of common respiratory tract is avoided.
Detection sensitivity: the constructed ORF1ab gene plasmid of 2019-nCOV virus was diluted in gradient at 108copies/ml、 107copies/ml、106copies/ml、105copies/ml、104copies/ml、103copies/ml、102copies/ml、101The detection sensitivity was verified using copies/ml, 1copies/ml, and 0copies/ml as templates, and the sensitivity detection results are shown in FIG. 8. As can be seen from FIG. 8, the detection sensitivity was 103copies/ml。
Detection of human reference gene
The reference gene is human CALR gene, AB385014.1
The sequence of the reference gene is shown in SEQ ID NO. 23.
The primers used for isothermal amplification of reference gene nucleic acid are shown in Table 10. The sequences of F3, B3, LF, LB, FIP and BIP of the primer for constant-temperature amplification of the internal reference gene nucleic acid are sequentially shown as SEQ ID NO. 24-SEQ ID NO. 29.
TABLE 10 primers for reference gene specific isothermal amplification
| Primer name | Primer sequences |
| F3 | ttgcagacaagccaggatg |
| B3 | agatgtcgggaccaaacatg |
| LF | gcctttgttgctgaaaggct |
| LB | cggctatgtgaagctgtttcc |
| FIP | tgaactgcaccaccagcgtcctctgtcggccagtttcg |
| BIP | gcagaacatcgactgtggggggtctgagtctccgtgcatgt |
The reaction system for isothermal amplification of reference gene nucleic acid is shown in Table 11.
TABLE 11 reaction System for isothermal amplification of reference Gene nucleic acids
And (3) amplification procedure: signals were collected once at 63 ℃ for 60min for 30 s.
Primer specificity: crossover experiments were performed and the detection of pathogens for crossover is shown in table 3.
The cross-reaction result is shown in fig. 9, and as can be seen from fig. 9, the specificity of detection is strong, and the cross-reaction is not caused with other pathogens of common respiratory tract.
Example 2
The LAMP combined detection method for the influenza A virus, the influenza B virus and the novel coronavirus comprises the following steps:
1) the nucleic acid of the purified sample is extracted by a conventional method.
2) Preparing a reaction system for isothermal amplification of influenza A virus nucleic acid, a reaction system for isothermal amplification of influenza B virus nucleic acid, a reaction system for isothermal amplification of 2019-nCOV virus nucleic acid (N gene), a reaction system for isothermal amplification of 2019-nCOV virus nucleic acid (ORF1ab gene) and a reaction system for isothermal amplification of internal reference CALR gene nucleic acid, performing loop-mediated isothermal amplification reaction, performing five groups of amplification reactions in an integrated device, performing fluorescence detection on the five groups of amplification reactions simultaneously, and performing signal collection on fluorescence change of a fluorescent dye SYBR Green I in a sample to form an amplification curve and display the amplification state of the sample so as to realize detection under the five reaction systems.
3) As a result, the threshold was set at 90 cycles, i.e., 45 minutes, and the number of positive cells below the threshold was positive and the number of negative cells above the threshold was negative.
Sequence listing
<110> Henan Zhi Tai Biotech Co., Ltd
Primer group, kit and method for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus
<160> 29
<170> SIPOSequenceListing 1.0
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<211> 1002
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tagatattga aagatgagtc ttctaaccga ggtcgaaacg tacgttctct ctatcgtccc 60
ttcaggcccc ctcaaagccg agatcgcgca gagacttgaa gatgtgtttg cagggaaaaa 120
caccgatctt gaggcgctca tggaatggct aaagacaaga ccaatcctgt cacctctgac 180
taaggggatt ttaggatttg tgttcacgct caccgtgccc agtgagcgag gactgcagcg 240
tagacgcttt gtccaaaatg cccttaatgg aaatggggat ccaaacaaca tggacagagc 300
ggtcaaactg tacaggaagc tcaagaggga aataacattc catggagcaa aagaggtggc 360
actcagttat tcaactggtg cacttgccag ctgcatgggc ctcatataca acaggatggg 420
gactgtgacc actgaagtgg catttggcct ggtgtgtgcc acgtgtgagc agattgctga 480
ttcccagcat cggtctcaca gacaaatggt gacaacaacc aacccgctaa tcagacatga 540
gaacaggatg gtactggcca gtactacggc taaggccatg gagcaaatgg cagggtcaag 600
tgagcaggca gcagaggcta tggaggttgc tagtcaggct aggcagatgg tgcaggcaat 660
gaggaccatt gggactcatc ctagctccag tgctggtctg aaagatgatc ttcttgaaaa 720
tttgcaggcc taccagaaac ggatgggagt gcaaatgcaa cgattcaagt gatcctctcg 780
ttattgccgc aagtatcatt gggatcttgc acttgatatt gtggattctt gatcgccttt 840
tcttcaaatg catttatcgt cgccttaaat acggtttgaa aagagggcct tctacggaag 900
gagtgcctga gtctatgagg gaggaatatc ggcaggaaca gcagagtgct gtggatgttg 960
acgatagtca ttttgtcaac atagagctgg agtaaaaaac ta 1002
<210> 2
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<213> Artificial Sequence (Artificial Sequence)
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tgtttagtca ctggcaaaca gaaaaaaaat ggcggacaat atgaccacaa cacaaattga 60
ggtgggtccg ggagcaacca atgccaccat aaacttcgaa gcaggaattc tggagtgcta 120
tgaaagactt tcatggcaaa gggcccttga ctaccctggt caagaccgtc taaacagact 180
aaagaggaaa ttagagtcaa gaataaagac ccacaacaaa agtgagcctg aaagtaaaag 240
aatgtctctt gaagagagaa aagcaattgg agtaaaaatg atgaaagtac ttctatttat 300
gaatccgtct gctggaattg aagggtttga gccatactgt atgaaaagtt cctcggagag 360
caactgtccg aaatacagtt ggattaatta cccttcaacc ccagggaggt gccttgatga 420
catagaagaa gaaccagagg atgttgatgg cccaactgaa atagtattaa gagacatgaa 480
caacagagat gcaaggcaaa gataaaggag gaagtaaaca ctcagaaaga agggaagttc 540
cgtttgacaa taagaaggga tatgcgtaat gtattgtccc tgagagtgtt agtaaacgga 600
acattcctca aacaccccaa tggatacaag tccttatcaa ctctacatag attgaatgca 660
tatgaccaga gtggaaggct tgttgctaaa cttgttgcta ctgatgatct tacagtggag 720
gatgaagaag atggccatcg gatcctcaat tcactcttcg agcgtcttaa tgaaggacat 780
ccaaagccaa ttcgagcagc tgaaactgcg gtgggagtct tatcccaatt tggtcaagag 840
caccgattat caccagaaga gggagacaat tagactggtc acggaagaac tttatctttt 900
aagtaaaaga attgatgata acatattgtt ccacaaaaca gtaatagcta acagctccat 960
aatagctgac atggttgtat cattatcatt attagaaaca ttgtatgaaa tgaaggatgt 1020
ggttgaagtg tacagcaggc agtgcttgtg aatttaaaat aaa 1063
<210> 3
<211> 599
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tctactacct aggaactggg ccagaagctg gacttcccta tggtgctaac aaagacggca 60
tcatatgggt tgcaactgag ggagccttga atacaccaaa agatcacatt ggcacccgca 120
atcctgctaa caatgctgca atcgtgctac aacttcctca aggaacaaca ttgccaaaag 180
gcttctacgc agaagggagc agaggcggca gtcaagcctc ttctcgttcc tcatcacgta 240
gtcgcaacag ttcaagaaat tcaactccag gcagcagtag gggaacttct cctgctagaa 300
tggctggcaa tggcggtgat gctgctcttg ctttgctgct gcttgacaga ttgaaccagc 360
ttgagagcaa aatgtctggt aaaggccaac aacaacaagg ccaaactgtc actaagaaat 420
ctgctgctga ggcttctaag aagcctcggc aaaaacgtac tgccactaaa gcatacaatg 480
taacacaagc tttcggcaga cgtggtccag aacaaaccca aggaaatttt ggggaccagg 540
aactaatcag acaaggaact gattacaaac attggccgca aattgcacaa tttgccccc 599
<210> 4
<211> 500
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggctgtag ttgtgatcaa ctccgcgaac ccatgcttca gtcagctgat gcacaatcgt 60
ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca ccgtgcggca caggcactag 120
tactgatgtc gtatacaggg cttttgacat ctacaatgat aaagtagctg gttttgctaa 180
attcctaaaa actaattgtt gtcgcttcca agaaaaggac gaagatgaca atttaattga 240
ttcttacttt gtagttaaga gacacacttt ctctaactac caacatgaag aaacaattta 300
taatttactt aaggattgtc cagctgttgc taaacatgac ttctttaagt ttagaataga 360
cggtgacatg gtaccacata tatcacgtca acgtcttact aaatacacaa tggcagacct 420
cgtctatgct ttaaggcatt ttgatgaagg taattgtgac acattaaaag aaatacttgt 480
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gcacacacca ggccaaat 18
<210> 7
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gccacctctt ttgctccatg gacatggaca gagcggtcaa ac 42
<210> 8
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tcaactggtg cacttgccag cccacttcag tggtcacagt c 41
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ttacccttca accccagg 18
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gttccgttta ctaacactct c 21
<210> 11
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gcatctttgt tgttcatgtc ccttagatga catagaagaa gaaccag 47
<210> 12
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
taaacactca gaaagaaggg aagttaggga caatacatta cgcata 46
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gccaaaaggc ttctacgca 19
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aatagcagtc cagatgacca aattg 25
<210> 16
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gcggtgatgc tgctcttg 18
<210> 17
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tcccctactg ctgcctggag gcagtcaagc ctcttctcg 39
<210> 18
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tctcctgcta gaatggctgg catctgtcaa gcagcagcaa ag 42
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
<210> 20
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
agtaagacgt tgacgtgat 19
<210> 21
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tggtagttag agaaagtgtg tctctgcttc caagaaaagg acgaa 45
<210> 22
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
taaggattgt ccagctgttg catgtggtac catgtcacc 39
<210> 23
<211> 500
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
acgggtggac ttcccgctgg atcgaatcca aacacaagtc agattttggc aaattcgttc 60
tcagttccgg caagttctac ggtgacgagg agaaagataa aggtttgcag acaagccagg 120
atgcacgctt ttatgctctg tcggccagtt tcgagccttt cagcaacaaa ggccagacgc 180
tggtggtgca gttcacggtg aaacatgagc agaacatcga ctgtgggggc ggctatgtga 240
agctgtttcc taatagtttg gaccagacag acatgcacgg agactcagaa tacaacatca 300
tgtttggtcc cgacatctgt ggccctggca ccaagaaggt tcatgtcatc ttcaactaca 360
agggcaagaa cgtgctgatc aacaaggaca tccgttgcaa ggatgatgag tttacacacc 420
tgtacacact gattgtgcgg ccagacaaca cctatgaggt gaagattgac aacagccagg 480
<210> 24
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ttgcagacaa gccaggatg 19
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
cggctatgtg aagctgtttc c 21
<210> 28
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tgaactgcac caccagcgtc ctctgtcggc cagtttcg 38
<210> 29
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gcagaacatc gactgtgggg ggtctgagtc tccgtgcatg t 41
Claims (9)
- A primer group for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus is characterized by comprising the following components: the influenza A virus amplification primer for detecting influenza A virus, the influenza B virus amplification primer for detecting influenza B virus and the 2019-nCOV virus amplification primer group for detecting novel coronavirus are disclosed, wherein the sequences of the influenza A virus amplification primer F3, B3, FIP and BIP are shown as SEQ ID NO:5 to SEQ ID NO:8, F3, B3, FIP and BIP sequences of the influenza B virus amplification primer are shown as SEQ ID NO:9 to SEQ ID NO:12, the 2019-nCOV virus amplification primer group consists of one or two groups of N gene amplification primers and ORF1ab gene amplification primers, wherein F3, B3, LF, LB, FIP and BIP sequences of the N gene primers are shown as SEQ ID NO:13 to SEQ ID NO:18, F3, B3, FIP and BIP sequences of the ORF1ab gene primer are shown as SEQ ID NO:19 to SEQ ID NO:22, respectively.
- The kit for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus, characterized by comprising the primer set for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus according to claim 1.
- 3. The kit for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus according to claim 2, characterized in that the addition amount ratio of F3, B3, FIP and BIP of the influenza A virus amplification primer is 0.5:0.5:4:4, the addition amount ratio of F3, B3, FIP and BIP of the influenza B virus amplification primer is 0.5:0.5:4:4, the addition amount ratio of F3, B3, LF, LB, FIP and BIP of the N gene primer is 0.5:0.5:1:1:4:4, and the addition amount ratio of F3, B3, FIP and BIP of the ORF1ab gene primer is 0.5:0.5:4: 4.
- 4. The kit for LAMP-linked detection of influenza A virus, influenza B virus and novel coronavirus according to claim 2 or 3, characterized by further comprising an internal reference gene and an internal reference gene amplification primer for detecting the internal reference gene.
- 5. The kit for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus according to claim 4, wherein the reference gene is a human CALR gene, and the sequence of the human CALR gene is shown in SEQ ID NO. 23; the sequences of F3, B3, LF, LB, FIP and BIP of the internal reference gene amplification primer are shown as SEQ ID NO. 24-SEQ ID NO. 29 in sequence.
- 6. The kit for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus according to claim 4, characterized by further comprising five groups of reaction systems corresponding to influenza A virus amplification primers, influenza B virus amplification primers, N gene amplification primers, ORF1ab gene amplification primers and reference gene amplification primers respectively, wherein each group of reaction systems comprises corresponding buffer solution and MgSO4Betaine, dNTPS, dUTP, chromogenic agent, Bst DNA polymerase, UNG enzyme and reverse transcriptase.
- 7. The LAMP combined detection kit for influenza A virus, influenza B virus and novel coronavirus according to claim 6, wherein each group of the reaction system has a volume of 23ul and comprises the following substances: 2.5ul of buffer solution, 0.75-1.5 ul of MgSO42-4 ul of Betaine, 2.5-3.75 ul of dNTPS, 1ul of dUTP, 0.5ul of color developing agent, 1ul of Bst DNA polymerase, 0.125ul of UNG enzyme and 0.1ul of reverse transcriptase, and further comprises the following primers: 0.5ul of F3, 0.5ul of B3, 0 or 1ul of LF, 0 or 1ul of LB, 4ul of FIP, 4ul of BIP.
- The LAMP combined detection method for the influenza A virus, the influenza B virus and the novel coronavirus is characterized by comprising the following steps of:1) extracting nucleic acid from the purified sample;2) preparing the LAMP reaction system in the kit of claim 2, and performing loop-mediated isothermal amplification reaction;3) and (6) judging a result.
- 9. The LAMP combined detection method for influenza A virus, influenza B virus and novel coronavirus according to claim 8, wherein amplification conditions are 60-65 ℃ and 30-60 min, and signals are collected for 30-60 s.
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| CN202011094570.3A CN112195275A (en) | 2020-10-14 | 2020-10-14 | Primer group, kit and method for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113073152A (en) * | 2021-05-12 | 2021-07-06 | 广州普世利华科技有限公司 | LAMP primer, probe and kit for detecting influenza B virus |
| CN113151585A (en) * | 2021-01-14 | 2021-07-23 | 复旦大学附属华山医院北院 | Primer composition and kit for rapidly detecting mutation of novel coronavirus D614G |
| CN113549710A (en) * | 2021-05-08 | 2021-10-26 | 弗罗朗(浙江)生物技术有限公司 | Kit for rapidly and specifically detecting 2019 novel coronavirus and use method thereof |
| CN113564279A (en) * | 2021-07-19 | 2021-10-29 | 广州达安基因股份有限公司 | Novel coronavirus, influenza A and B virus detection kit and method |
| CN113584221A (en) * | 2021-05-08 | 2021-11-02 | 弗罗朗(浙江)生物技术有限公司 | Kit for rapidly and specifically detecting influenza A virus and use method thereof |
| CN113832259A (en) * | 2021-08-26 | 2021-12-24 | 中国科学院青岛生物能源与过程研究所 | Novel rapid detection kit for coronavirus and application thereof |
| CN114015810A (en) * | 2021-12-03 | 2022-02-08 | 浙江大学 | Detection kit for simultaneous detection of coronavirus RNA double genes and identification of mutant strains |
| CN114277192A (en) * | 2021-12-30 | 2022-04-05 | 南京巨匠生物科技有限公司 | Amplification system composition for RT-LAMP reaction, RT-LAMP method of RNA virus and application |
| EP4056718A1 (en) * | 2021-03-12 | 2022-09-14 | Industry-Academic Cooperation Foundation, Chosun University | Composition for detection of sars-cov-2 virus gene and covid-19 diagnosismethod using real-time rt-pcr |
| CN115852049A (en) * | 2022-10-27 | 2023-03-28 | 广州海关技术中心 | Kit for detecting neocorona, respiratory syncytial, influenza and respiratory adenovirus and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164538A (en) * | 2017-07-11 | 2017-09-15 | 复旦大学附属华山医院 | A kind of the internal reference amplimer composition and its amplification system of detection CALR gene mutations |
| KR20200056831A (en) * | 2018-11-15 | 2020-05-25 | 고려대학교 산학협력단 | The primer set for detecting Influenza virus, a kit comprising the same, and detection method using the same |
| CN111411172A (en) * | 2020-02-28 | 2020-07-14 | 江苏硕世生物科技股份有限公司 | Probe and primer composition for simultaneously detecting novel human coronavirus, influenza A virus and influenza B virus |
| CN111549183A (en) * | 2020-06-08 | 2020-08-18 | 国药集团武汉血液制品有限公司 | Method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of convalescent coronary pneumonia convalescent patient |
-
2020
- 2020-10-14 CN CN202011094570.3A patent/CN112195275A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164538A (en) * | 2017-07-11 | 2017-09-15 | 复旦大学附属华山医院 | A kind of the internal reference amplimer composition and its amplification system of detection CALR gene mutations |
| KR20200056831A (en) * | 2018-11-15 | 2020-05-25 | 고려대학교 산학협력단 | The primer set for detecting Influenza virus, a kit comprising the same, and detection method using the same |
| CN111411172A (en) * | 2020-02-28 | 2020-07-14 | 江苏硕世生物科技股份有限公司 | Probe and primer composition for simultaneously detecting novel human coronavirus, influenza A virus and influenza B virus |
| CN111549183A (en) * | 2020-06-08 | 2020-08-18 | 国药集团武汉血液制品有限公司 | Method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of convalescent coronary pneumonia convalescent patient |
Non-Patent Citations (5)
| Title |
|---|
| GUN-SOO PARK等: ""Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)"", 《THE JOURNAL OF MOLECULAR DIAGNOSTICS》 * |
| 崔淑娟等: ""甲型流感病毒逆转录环介导等温扩增快速检测方法的建立"", 《中国预防医学杂志》 * |
| 王瑞丽: ""基于微流控等温扩增技术的流感病毒快速检测方法研究"", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 蔡欣等: ""流感病毒诊断方法的研究进展"", 《医学研究杂志》 * |
| 谭贵良等: "《现代分子生物学及组学技术在食品安全检测中的应用》", 30 June 2014, 中山大学出版社 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113151585A (en) * | 2021-01-14 | 2021-07-23 | 复旦大学附属华山医院北院 | Primer composition and kit for rapidly detecting mutation of novel coronavirus D614G |
| EP4056718A1 (en) * | 2021-03-12 | 2022-09-14 | Industry-Academic Cooperation Foundation, Chosun University | Composition for detection of sars-cov-2 virus gene and covid-19 diagnosismethod using real-time rt-pcr |
| CN113549710A (en) * | 2021-05-08 | 2021-10-26 | 弗罗朗(浙江)生物技术有限公司 | Kit for rapidly and specifically detecting 2019 novel coronavirus and use method thereof |
| CN113584221A (en) * | 2021-05-08 | 2021-11-02 | 弗罗朗(浙江)生物技术有限公司 | Kit for rapidly and specifically detecting influenza A virus and use method thereof |
| CN113073152A (en) * | 2021-05-12 | 2021-07-06 | 广州普世利华科技有限公司 | LAMP primer, probe and kit for detecting influenza B virus |
| CN113564279A (en) * | 2021-07-19 | 2021-10-29 | 广州达安基因股份有限公司 | Novel coronavirus, influenza A and B virus detection kit and method |
| CN113832259A (en) * | 2021-08-26 | 2021-12-24 | 中国科学院青岛生物能源与过程研究所 | Novel rapid detection kit for coronavirus and application thereof |
| CN114015810A (en) * | 2021-12-03 | 2022-02-08 | 浙江大学 | Detection kit for simultaneous detection of coronavirus RNA double genes and identification of mutant strains |
| CN114277192A (en) * | 2021-12-30 | 2022-04-05 | 南京巨匠生物科技有限公司 | Amplification system composition for RT-LAMP reaction, RT-LAMP method of RNA virus and application |
| CN114277192B (en) * | 2021-12-30 | 2022-11-18 | 南京巨匠生物科技有限公司 | Amplification system composition for RT-LAMP reaction, RT-LAMP method of RNA virus and application |
| CN115852049A (en) * | 2022-10-27 | 2023-03-28 | 广州海关技术中心 | Kit for detecting neocorona, respiratory syncytial, influenza and respiratory adenovirus and application thereof |
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