CN111733287B - Kit for detecting pathogenic nucleic acid of fever with thrombocytopenia syndrome - Google Patents
Kit for detecting pathogenic nucleic acid of fever with thrombocytopenia syndrome Download PDFInfo
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- CN111733287B CN111733287B CN202010573257.1A CN202010573257A CN111733287B CN 111733287 B CN111733287 B CN 111733287B CN 202010573257 A CN202010573257 A CN 202010573257A CN 111733287 B CN111733287 B CN 111733287B
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- 206010037660 Pyrexia Diseases 0.000 title claims abstract description 30
- 206010043554 thrombocytopenia Diseases 0.000 title claims abstract description 29
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Abstract
The invention discloses a kit for detecting pathogenic nucleic acid of fever with thrombocytopenia syndrome, which comprises: crRNA-1, lbCas12a nuclease for detecting pathogenic nucleic acid of fever with thrombocytopenia syndrome a nucleic acid detection probe, a first upstream primer a first downstream primer,
DNA Amplification kit, water and 1 XNEbuffer TM 2.1; the nucleic acid detection probe is IAB-TTATT-BHQ-1; the nucleotide sequence of the first upstream primer is shown as SEQ ID NO. 3; the nucleotide sequence of the first downstream primer is shown as SEQ ID NO. 4. The kit can quickly and accurately detect the SFTS pathogen nucleic acid, and has the advantages of good detection specificity, high sensitivity, realization of high-sensitivity detection at room temperature, and convenient and quick operation.
Description
Technical Field
The invention belongs to the technical field of molecular biology. More particularly, it relates to a kit for detecting nucleic acids of pathogens of the fever with thrombocytopenia syndrome (SFTS).
Background
The main clinical manifestations of fever with thrombocytopenia syndrome (SFTS) pathogen infected persons are fever, digestive tract symptoms, thrombocytopenia, leukopenia, liver and kidney function damage, and bleeding manifestations of some infected persons. The disease mainly occurs in hills and mountainous areas, and patients mainly take adult farmers who engage in agricultural production, and part of the patients are bitten by ticks. The commonly used diagnostic methods are often based on empirical judgment or detection of individual pathogens, and products based on global detection of common pathogens of syndrome are rare. In order to realize the early detection of the SFTSV, it is very important to develop a diagnostic method for accurately, efficiently and quickly detecting the pathogen of the SFTS with low cost.
At present, in the detection of pathogens with fever accompanied by thrombocytopenia (SFTS), the gold standard is still a Real-time fluorescence quantitative PCR (RT-PCR) detection technology, and the sensitivity and the specificity are higher. Real-time fluorescent PCR detection alone, which generally takes about two hours, is used. The whole detection process also comprises the steps of sampling, processing samples and the like, and the total time is usually more than two hours. The method has the main disadvantages that the detection takes long time, and the PCR detection needs to depend on a PCR instrument or an expensive real-time quantitative PCR instrument and other various matched equipment.
Therefore, a highly sensitive kit that does not require expensive equipment, is low cost, and can rapidly and accurately generate fever with thrombocytopenia syndrome (SFTS) pathogenic viruses is highly desired.
Disclosure of Invention
The object of the present invention is to overcome the disadvantages of the prior art and to provide crRNA-1 for detecting the nucleic acid of the causative agent of fever with thrombocytopenia syndrome.
It is a second object of the present invention to provide crRNA-2 for detecting nucleic acids of pathogens of fever with thrombocytopenia syndrome.
It is a third object of the present invention to provide a kit for detecting a nucleic acid of a causative agent of fever with thrombocytopenia syndrome.
It is a fourth object of the present invention to provide a second kit for detecting a nucleic acid of a causative agent of fever with thrombocytopenia syndrome.
The technical scheme of the invention is summarized as follows:
the method is used for detecting the crRNA-1 of the pathogenic nucleic acid of the fever with thrombocytopenia syndrome, and the nucleotide sequence of the crRNA-1 is shown as SEQ ID NO. 1.
Detecting the crRNA-2 of the pathogenic nucleic acid of the fever with thrombocytopenia syndrome, wherein the nucleotide sequence of the crRNA-2 is shown as SEQ ID NO. 2.
The first kit for detecting pathogenic nucleic acid of the fever with thrombocytopenia syndrome comprises crRNA-1, lbCas12a nuclease, a nucleic acid detection probe, a first upstream primer, a first downstream primer, a second upstream primer and a second downstream primer which are used for detecting the pathogenic nucleic acid of the fever with thrombocytopenia syndrome,
DNA Amplification kit, water and 1 XNEbuffer TM 2.1；
The nucleic acid detection probe is IAB-TTATT-BHQ-1;
the nucleotide sequence of the first upstream primer is shown as SEQ ID NO. 3;
the nucleotide sequence of the first downstream primer is shown as SEQ ID NO. 4;
the second kit for detecting pathogenic nucleic acid of the fever with thrombocytopenia syndrome comprises crRNA-2, lbCas12a nuclease, a nucleic acid detection probe, a second upstream primer, a second downstream primer, a probe for detecting the pathogenic nucleic acid of the fever with thrombocytopenia syndrome,
DNA Amplification kit, water and 1 XNEbuffer TM 2.1；
The nucleic acid detection probe is IAB-TTATT-BHQ-1;
the nucleotide sequence of the second upstream primer is shown as SEQ ID NO. 5;
the nucleotide sequence of the second downstream primer is shown as SEQ ID NO. 6;
the invention has the advantages that:
the kit for detecting the pathogen nucleic acid of the fever with thrombocytopenia syndrome (SFTS) can quickly and accurately detect the pathogen nucleic acid of the SFTS, and has the advantages of good detection specificity, high sensitivity, realization of high-sensitivity detection at room temperature, and convenient and quick operation.
Drawings
FIG. 1 is a graph showing the results of detection of SFTS pathogen nucleic acid L fragments mock samples (plasmids) using the kit of example 2.
FIG. 2 is a graph showing the results of detecting SFTS pathogen nucleic acid L fragment mock samples (plasmids) using the kit of example 3.
Detailed Description
The present invention will be further illustrated by the following specific examples.
Example 1
crRNA designed to detect the nucleic acid of the causative agent of fever with thrombocytopenia syndrome (SFTS).
And acquiring a genome sequence of the SFTS pathogen, and searching a specific identification region of the SFTS pathogen through bioinformatics analysis and comparison.
The specific operation steps are as follows, searching the whole genome sequence of the existing SFTS pathogen HB-29 in the laboratory in the NCBI nucleic acid sequence library to obtain the whole genome sequence. Sequences of "TTTN" were searched for within the HB-29 whole genome sequence and used as an alternative database for crRNA targeting sequences.
According to the screening principle, parameters such as GC content of the sequence, base uniformity, sequence conservation and the like are obtained, a target site recognition sequence with the size of 20-24nt is finally obtained, then a direct repetitive sequence with the size of 20-21nt is added with the obtained target site recognition sequence with the size of 20-24nt to obtain the crRNA for detecting SFTS pathogen nucleic acid, 2 pieces are obtained in total, the nucleotide sequences of the crRNA-1 and the crRNA-2 are respectively shown as SEQ ID NO.1, and the nucleotide sequence of the crRNA-2 is shown as SEQ ID NO. 2.
Example 2
A kit for detecting a nucleic acid of a causative agent of fever with thrombocytopenia syndrome (SFTS), comprising: crRNA-1, lbCas12a nucleases for detection of SFTS pathogen nucleic acids (A) ((B))
Lba Cas12a (Cpf 1), bioLabs), nucleic acid detection probe, first upstream primer, first downstream primer, and,
DNA Amplification kit (commercially available), water (DEPC-treated water) and 1 XNEbuffer TM 2.1(BioLabs)；
The nucleic acid detection probe is IAB-TTATT-BHQ-1 (manufactured by consignment of biological companies);
the nucleotide sequence of the first upstream primer is shown as SEQ ID NO. 3;
the nucleotide sequence of the first downstream primer is shown as SEQ ID NO. 4.
Example 3
A second kit for detecting a nucleic acid of a causative agent of fever with thrombocytopenia syndrome (SFTS), comprising: crRNA-2, lbCas12a nucleases for detection of SFTS pathogen nucleic acids: (
Lba Cas12a (Cpf 1), bioLabs), nucleic acid detection probe, second forward primer, second reverse primer,
DNA Amplification kit (commercially available), water (DEPC-treated water) and 1 XNEbuffer TM 2.1(BioLabs)；
The nucleic acid detection probe is IAB-TTATT-BHQ-1 (manufactured by entrusted biological companies);
the nucleotide sequence of the second upstream primer is shown as SEQ ID NO. 5;
the nucleotide sequence of the second downstream primer is shown as SEQ ID NO. 6.
Example 4
Use of the kit of example 2:
(1) A first upstream primer aqueous solution having a concentration of 2.4uL of 10. Mu.M, a first downstream primer aqueous solution having a concentration of 2.4uL of 10. Mu.M, and a first upstream primer aqueous solution having a concentration of 4uL of 1.8x10 -5 ng/uL detection simulation sample water solution (SFTS pathogen nucleic acid L fragment genome nucleotide sequence is shown in SEQ ID NO.7, GENBANK gene sequence number is KP 202165.1) is added
In the DNA Amplification mixing System: (
The DNA Amplification mixing system is
Preparing each component of the kit of DNA Amplification according to the instruction), and amplifying for 20min at 39 ℃;
(2) 1uL LbCas12a nuclease and 3uL1 XNEbuffer TM 2.1 and 4uL of crRNA-1 aqueous solution with the concentration of 1 mu M for detecting SFTS pathogen nucleic acid are incubated, and the temperature is 25 ℃ for 10min;
(3) 20uL of the amplification product obtained in step (1) and 8uL of the incubation product obtained in step (2) were mixed in a 384-well plate, and then 2uL of an aqueous solution of a nucleic acid detecting probe at a concentration of 1. Mu.M was added. The reaction system is placed in a multifunctional microplate reader, the reaction is carried out for 20min at 37 ℃, and the fluorescence value is taken for result interpretation.
And (3) judging effective results: after removing background fluorescence of the sample, the sample is defined as positive when the fluorescence value is more than or equal to 3 times of that of the negative control sample, and the sample is defined as negative when the fluorescence value is less than 3 times of that of the negative control sample. See fig. 1.
FIG. 1 is a graph showing the results of detection of SFTS pathogen nucleic acid L fragments mock samples (plasmids) using the kit of example 2.
Example 5
Use of the kit of example 3:
(1) A second upstream primer aqueous solution having a concentration of 2.4uL of 10. Mu.M, a second downstream primer aqueous solution having a concentration of 2.4uL of 10. Mu.M, and 4uL of 1.8x10 -5 ng/uL detection simulation sample water solution (SFTS pathogen nucleic acid L fragment genome nucleotide sequence is shown in SEQ ID NO.7, GENBANK gene sequence number is KP 202165.1) is added
In the DNA Amplification mixing System: (
The DNA Amplification mixing system is
Preparing each component of the kit of the DNA Amplification according to the instruction), and amplifying for 20min at 39 ℃;
(2) 1uL LbCas12a nuclease and 3uL1 XNEbuffer TM 2.1 and 4uL of crRNA-2 aqueous solution with the concentration of 1 mu M for detecting SFTS pathogen nucleic acid are incubated, and the temperature is 25 ℃ for 10min;
(3) 20uL of the amplification product obtained in step (1) and 8uL of the incubation product obtained in step (2) were mixed in a 384-well plate, and then 2uL of an aqueous solution of a nucleic acid detecting probe at a concentration of 1. Mu.M was added. The reaction system is placed in a multifunctional microplate reader, the reaction is carried out for 20min at 37 ℃, and the fluorescence value is taken for result interpretation.
And (3) judging effective results: after removing background fluorescence of the sample, the sample is defined as positive when the fluorescence value is more than or equal to 3 times of that of the negative control sample, and the sample is defined as negative when the fluorescence value is less than 3 times of that of the negative control sample. See fig. 2.
FIG. 2 is a graph showing the results of detection of SFTS pathogen nucleic acid L fragments mock samples (plasmids) using the kit of example 3.
Sequence listing
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caagaacaaa gaggagtcaa cagagctaaa tgccctcgga gaaatgtaca agaagattgt 2340
ggagatggaa gaggacaagc ccagtagccc tgagtttcta gggtggggag acacagactc 2400
ccctaagaag catgaattct cacggagctt cctcagagct gcttgctcat ctctggagag 2460
agaaattgct cagcggcatg gaagacaatg gaagcagaac cttgaggagc gtgtcctgaa 2520
agagattggg accaagaaca tcctggacct tgcatccatg aaagctacaa gcaacttttc 2580
caaagattgg gagctctact cagaagtcca gaccaaagag taccataggt ccaaactgct 2640
ggagaagatg gccacattga ttgagaaggg ggttatgtgg tacattgatg ctgtgggcca 2700
ggcatggaag gcagttctgg atgacgggtg catgcgaatc tgtctcttca aaaagaatca 2760
gcatggtggc ctcagagaga tctacgttat ggatgctaat gcccggcttg tgcagtttgg 2820
ggtcgagacc atggccaggt gtgtctgtga gctaagccca catgagactg ttgccaaccc 2880
taggctcaag aattctatca tagagaacca tgggctgaag tcagcccgca gtcttggccc 2940
tggctctatt aacataaact catccaatga tgccaagaag tggaatcagg ggcactacac 3000
aacaaagcta gctctagttc tttgttggtt catgccagcc aaattccaca gattcatttg 3060
ggctgccatt tccatgtttc ggagaaaaaa gatgatggtg gacctaaggt ttttggctca 3120
cctcagtact aaatctgagt ctaggtcatc tgatccgttc agggaagcaa tgacagacgc 3180
cttccatggt aatagggaag tttcatggat ggacaaaggg cgaacttaca taaagacaga 3240
gacaggaatg atgcagggca tactgcactt tacatctagc ctcctccact cttgtgttca 3300
gagcttctac aagtcctatt tcgtctcgaa gctcaaggag ggctacatgg gggaaagcat 3360
cagtggggtg gtggatgtca tagaaggctc tgacgactca gcgatcatga tcagcatacg 3420
ccccaagtca gacatggatg aagtccgatc aaggttcttt gttgctaact tgctccactc 3480
tgtcaagttc ttgaaccctt tgtttgggat ttattcttca gagaaatcaa cagtgaacac 3540
agtgtattgt gtcgagtata actctgaatt ccatttccac aggcacttgg ttagacccac 3600
actgagatgg atagcagcgt ctcaccaaat ctcagagaca gaagcccttg caagcaggca 3660
agaagattac tccaaccttc taacgcagtg cttggaagga ggggcctcat tctctcttac 3720
ctacctcata cagtgcgctc agctcctgca ccactacatg cttctaggac tatgcttaca 3780
tcccttattt ggaaccttca tggggatgtt gatatcagac ccagatccag ccctagggtt 3840
cttcctcatg gacaaccctg cattcgcagg gggtgcagga tttagattca atctgtggag 3900
agcctgcaag actacagacc ttggacggaa gtatgcttat tattttaatg agatacaggg 3960
taaaacaaag ggagatgagg actacagagc tctggatgcc acatcgggag gaactctcag 4020
ccactctgtt atggtgtatt ggggggacag gaagaagtat caggctttgt tgaacaggat 4080
gggccttcct gaggactggg tagagcagat agatgagaat cctggagtcc tttacaggag 4140
agctgccaac aagaaggaac tactcttaaa actggcagag aaggttcatt ctcctggtgt 4200
aactagcagc ctgagtaagg ggcatgtggt gcctcgggtg gtggcagcag gagtatacct 4260
tctctcacgc cactgctttc gctttagctc aagcattcat ggcaggggct cagcacagaa 4320
ggctagcctt ataaaactgc taatgatgtc ttctgtttct gccatgaagc acgggggctc 4380
actaaaccct aatcaggagc gaatgctctt ccctcaggct caagagtatg acagagtatg 4440
cacattgctt gaggaagttg aacacctaac agggaaattt gttgttaggg agagaaacat 4500
tgtcaggagc cgcatagact tgttccaaga accagtggac ttgcggtgca aggcagagga 4560
tctggtgtca gaggtgtggt ttggcctgaa aaggactaag cttggacccc gtctcctcaa 4620
ggaagagtgg gacaaactta gggcttcatt tgcatggctg agcacagacc catctgaaac 4680
actgagggat ggtccttttc ttagccatgt gcagtttagg aacttcatag cccacgttga 4740
tgccaaatca agatcagtca ggctcctggg tgcccccgtg aagaagtcag gtggggtcac 4800
caccataagc caagtagtta gaatgaactt cttccctggt tttagcctag aagctgagaa 4860
gagcttagat aatcaggaaa gacttgagag catctccatc ctcaagcatg tcttgttcat 4920
ggtcttgaat ggcccataca ctgaggagta caagctggag atgatcatag aggccttctc 4980
tactcttgtg ataccacagc catcagaggt catcaggaaa tctaggacca tgaccttatg 5040
cctcttatcg aattacttgt ctagtaaggg tgggtccatt ctagaccaga ttgagagggc 5100
acagtcagga actctaggag gctttagcaa gccccagaag acattcatta ggccaggagg 5160
tggtgttggc tataagggaa aaggtgtgtg gactggggtg atggaggaca cccatgttca 5220
aattctgata gatggagatg ggactagtaa ctggcttgag gagatcaggc tcagtagtga 5280
tgccaggctt tatgatgtca ttgaatccat ccgaaggtta tgtgatgacc ttgggatcaa 5340
caacagggtg gcatctgcat atagaggtca ttgcatggtt aggctgagtg gattcaagat 5400
caagccagca tcaaggactg acgggtgtcc agtcaggatc atggaaaggg gcttcaggat 5460
tagggaactt caaaatccgg atgaggtcaa gatgagagtg aggggcgaca tcctcaacct 5520
ctctgtcaca atacaagaag gaagggtcat gaacattctg agctacaggc caagagacac 5580
tgatatatca gagtcagctg cagcatacct ctggagtaat cgagacctct tctcctttgg 5640
gaagaaggaa ccatcctgca gctggatctg cttaaaaact cttgacaatt gggcctggtc 5700
acatgcctca gttctcctgg caaatgatag gaagacccaa ggcattgaca atagagctat 5760
ggggaacatt ttcagggact gtcttgaagg ttctcttaga aagcaagggc tgatgaggtc 5820
aaagctcaca gagatggtgg agaagaatgt agttccttta acaactcaag agctcgtcga 5880
catcctggag gaggacattg acttttcaga tgtcatagct gtggagctct cagaggggtc 5940
gcttgacatt gaatccatct ttgatggggc acctatcttg tggtctgctg aggtggaaga 6000
gtttggagaa ggagtggtgg ctgtgagcta ttccagtaag tactatcatc taaccctgat 6060
ggaccaagct gccatcacaa tgtgtgcgat catgggtaag gaaggctgta gagggcttct 6120
taccgagaag agatgcatgg cagccatacg agagcaggta cggccattcc tcatattcct 6180
gcaaatacct gaggacagca tttcttgggt gtctgatcag ttctgcgact ccaggggcct 6240
tgatgaagag agcaccatta tgtggggtta actttgaaac acggttggaa cgcagttgat 6300
gtgtctgtgg gtggctaggg agtgttggtt ttagaaggat ttccttaaga tctgggcggt 6360
ctttgtgt 6368
Claims (4)
1. The crRNA-1 for detecting the nucleic acid of the pathogenic agent of the fever with thrombocytopenia syndrome is characterized by being shown as SEQ ID NO. 1.
2. The crRNA-2 for detecting the nucleic acid of the pathogenic agent of the fever with thrombocytopenia syndrome is characterized by being shown as SEQ ID NO. 2.
3. A kit for detecting a nucleic acid of a pathogenic agent of fever with thrombocytopenia syndrome, which is characterized by comprising: device for detecting fever with thrombocytopeniacrRNA-1, lbCas12a nucleases, which characterize pathogen nucleic acids a nucleic acid detection probe, a first upstream primer a first downstream primer,
DNA Amplification kit, water and 1 XNEbuffer TM 2.1；
The nucleic acid detection probe is IAB-TTATT-BHQ-1;
the nucleotide sequence of the first upstream primer is shown as SEQ ID NO. 3;
the nucleotide sequence of the first downstream primer is shown as SEQ ID NO. 4.
4. The kit for detecting the nucleic acid of the pathogenic agent of the fever with thrombocytopenia syndrome is characterized by comprising: crRNA-2, lbCas12a nuclease, a nucleic acid detection probe, a second upstream primer, a second downstream primer, a nucleic acid for detecting pathogenic nucleic acid of fever with thrombocytopenia syndrome,
DNA Amplification kit, water and 1 XNEbuffer TM 2.1；
The nucleic acid detection probe is IAB-TTATT-BHQ-1;
the nucleotide sequence of the second upstream primer is shown as SEQ ID NO. 5;
the nucleotide sequence of the second downstream primer is shown as SEQ ID NO. 6.
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| CN113755646A (en) * | 2021-10-09 | 2021-12-07 | 安徽医科大学第四附属医院 | CrRNA and CRISPR-Cas12a system for novel bunyavirus detection and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154585A (en) * | 2015-07-14 | 2015-12-16 | 江苏省疾病预防控制中心 | Isothermal amplification reagent kit for detecting fever and thrombocytopenia syndrome viruses and application of isothermal amplification reagent kit |
| WO2019011022A1 (en) * | 2017-07-14 | 2019-01-17 | 上海吐露港生物科技有限公司 | Application of cas protein, method for detecting target nucleic acid molecule and kit |
| CN111077310A (en) * | 2019-12-04 | 2020-04-28 | 天津大学 | Enzyme linked immunosorbent assay kit for detecting fever with thrombocytopenia syndrome virus antigen |
| CN111108220A (en) * | 2017-03-15 | 2020-05-05 | 博德研究所 | CRISPR-Effector System-based diagnostics for Virus detection |
| CN111254223A (en) * | 2020-03-21 | 2020-06-09 | 上海海关动植物与食品检验检疫技术中心 | Reaction system and kit for detecting African swine fever virus nucleic acid and application of reaction system and kit |
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| US11021740B2 (en) * | 2017-03-15 | 2021-06-01 | The Broad Institute, Inc. | Devices for CRISPR effector system based diagnostics |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154585A (en) * | 2015-07-14 | 2015-12-16 | 江苏省疾病预防控制中心 | Isothermal amplification reagent kit for detecting fever and thrombocytopenia syndrome viruses and application of isothermal amplification reagent kit |
| CN111108220A (en) * | 2017-03-15 | 2020-05-05 | 博德研究所 | CRISPR-Effector System-based diagnostics for Virus detection |
| WO2019011022A1 (en) * | 2017-07-14 | 2019-01-17 | 上海吐露港生物科技有限公司 | Application of cas protein, method for detecting target nucleic acid molecule and kit |
| CN111077310A (en) * | 2019-12-04 | 2020-04-28 | 天津大学 | Enzyme linked immunosorbent assay kit for detecting fever with thrombocytopenia syndrome virus antigen |
| CN111254223A (en) * | 2020-03-21 | 2020-06-09 | 上海海关动植物与食品检验检疫技术中心 | Reaction system and kit for detecting African swine fever virus nucleic acid and application of reaction system and kit |
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