CN101603096A - A kind of method and test kit that detects infectious disease pathogens - Google Patents
A kind of method and test kit that detects infectious disease pathogens Download PDFInfo
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Abstract
The present invention discloses a kind of method that the infectious disease pathogens that may be present in the biological sample are detected, described infectious disease pathogens comprise avian influenza virus H5 hypotype, avian influenza virus H7 hypotype, sars coronavirus, Hantaan virus, Ye Shi plague bacillus and anthrax bacillus, comprising: the nucleic acid fragment of amplification biological sample and detect with probe.The present invention also is provided for primer that increases and the probe that is used to detect.The present invention also provides the test kit that comprises above-mentioned primer.The method of the invention is highly sensitive, and high specificity is simple to operate, and sample range is wide, can detect multiple infectious disease pathogens simultaneously, is applicable to the early diagnosis of respiratory infectious disease.
Description
Technical field
The present invention relates to molecular biology, particularly relate to the method and the test kit that detect infectious disease pathogens.
Background technology
At the initial stage of disease transmission, accurately, fast, the pathogenic agent of transmissible disease detects easily, be the key of control disease transmission.Though set up the Monitoring systems of transmissible disease on internal and international, existing detection means respectively has limitation.Serological testing utilizes corresponding antibody to combine with antigen and detects pathogenic agent, this method diagnosis fast, simply, easy handling, detect but must prepare, especially under the situation that does not also have antibody sources, be not suitable for the early diagnosis of transmissible disease at obtaining behind the antibody of this pathogenic agent.
The pathogen isolation method is by directly pathogenic agent being carried out separation and Culture, detect identifying pathogenic agent.Highly sensitive, the high specificity of this method, but complicated operation, time-consuming (will spend week age at least), very high to laboratory Biosafety conditional request, thus be difficult in epidemic situation generation field by using, and also not every pathogenic agent can both obtain by the pathogen isolation method.
Nucleic acid detection technique is easy and simple to handle, and reaction is quick, especially the real-time fluorescence quantitative PCR of development in recent years (Real time PCR), highly sensitive, and can monitor the entire reaction process, but exist DNA to pollute the high problem of false positive rate that causes always, and because its threshold value is not obvious, therefore detected result gray area broad can't stdn, and because instrument requires high, have high input, operator are required height, need carry out the professional skill training, also limited the application of multiple detection.This method still can't detect for the extremely low sample of pathogenic agent content simultaneously.
NASBA (Nucleic acid sequence-based amplification, NASBA rely on the amplification technique of nucleotide sequence) be one continuously, isothermal, based on the nucleic acid amplification technologies of enzyme reaction.The reaction system of this technology comprises ThermoScript II, ribonuclease H, phage t7 ribonucleic acid polymerase and two specially designed Oligonucleolide primers, its upstream primer 3 ' end and 3 of template ' terminal complementation, 5 ' end contains the promoter sequence of the RNA polymerase that depends on DNA of phage t7, downstream primer 3 ' end sequence is consistent with 5 ' end sequence of template, and 5 ' end contains and capture probe complementary sequence.At the amplification initial period, upstream primer synthesizes and target RNA complementary Antisense cDNA under the effect of ThermoScript II with after just RNA template combine, and originally RNA template is then degraded by RnaseH.Then downstream primer and cDNA hybridization, synthetic double chain cDNA under the effect of the archaeal dna polymerase characteristic of ThermoScript II, promptly the double-stranded cDNA of corresponding target RNA copies.Because double-stranded cDNA one end includes the promoter sequence of T7RNA polymerase, thereby induced the activity of RNA polymerase, synthetic a large amount of and target sequence complementary sense-rna chain.So circulate repeatedly, the RNA copy number is constantly amplified.Simultaneously because another end of double-stranded cDNA has also been integrated and capture probe complementary sequence, therefore the complementary RNA that is produced again can be special in conjunction with capture probe, to be used for next step detection.
It is that the NASBA amplified production combines with capture probe one end at first specifically that enzyme connects oligonucleotide capture technique principle, the latter's the other end can be fixedly connected on the microwell plate, add pathogen specific detection probes and reaction substrate then, detect the ratio chrominance signal that produces, this detection reading is directly proportional with the amplified production amount of RNA, detect NASBA amplified production total amount with this, judge the infection conditions of viral template.This method is simple to operate, and high specificity is applicable to the rapid detection of a large amount of samples.
Transmissible disease of Chu Xianing such as respiratory infectious diseases such as SARS, bird flu in recent years, velocity of propagation is fast, case fatality rate is high, very big to social danger.The respiratory infectious disease common symptoms that avian influenza virus, sars coronavirus, Hantaan virus, Ye Shi plague bacillus, anthrax bacillus cause is fever, headache, also can be with feeling sick, vomitting, the pathogenic height of these pathogenic agent and be difficult to distinguish, the very easily best moment of delay diagnosis and treatment.Have not yet to see the detection method that report can detect and differentiate simultaneously above-mentioned six kinds of infectious disease pathogens, also do not have corresponding clinical detection reagent box listing.
Summary of the invention
The present invention seeks to overcome the defective that prior art can not detect multiple infectious disease pathogens simultaneously, a kind of method that the infectious disease pathogens that may be present in the biological sample are detected is provided, described infectious disease pathogens comprise avian influenza virus H5 hypotype, avian influenza virus H7 hypotype, sars coronavirus, Hantaan virus and Ye Shi plague bacillus, comprising:
(i) nucleic acid fragment of amplification biological sample, described nucleic acid fragment is the combination of following one or more:
The nucleic acid fragment of avian influenza virus H5 hypotype HA gene shown in SEQ ID NO.1;
The nucleic acid fragment of avian influenza virus H7 hypotype HA gene shown in SEQ ID NO.2;
The nucleic acid fragment of the nucleocapsid protein gene of sars coronavirus shown in SEQ ID NO.3;
The nucleic acid fragment of Hantaan virus nucleocapsid protein gene shown in SEQ ID NO.4;
The nucleic acid fragment of Ye Shi plague bacillus pPCP1 gene shown in SEQ ID NO.5;
The nucleic acid fragment of anthrax bacillus putative protein gene shown in SEQ ID NO.6;
(ii) detect a kind of specific hybrid in described probe and step (i) amplified production with probe.
A preferred embodiment of the present invention is the nucleic acid fragment that described step (i) is utilized NASBA technology amplification sample.The preferred operational conditions of NASBA is 41 ℃～45 ℃ incubations 90～150 minutes.
Step of the present invention (ii) in the hybridization of preferably described probe and step (i) gained amplified production on solid support, carry out.
Can stationary probe and one or more of nucleic acid fragment, for example, be fixed on the Sptting plate.Term " solid support " is meant and keeps under its hybridization characteristic prerequisite the oligonucleotide probe can the link coupled solid substrate that usually, solid substrate is nylon end, cellulose membrane, microballon, chip or Sptting plate.Before fixing, can modify probe, to promote fixing or to improve hybridization efficiency.Such modification comprises homopolymeric tailing, with NH
2Group or vitamin H, hapten conjugation.
Perhaps, probe and nucleic acid fragment all are unfixed, and hybridization can be carried out in liquid medium, and its detection can be carried out with flow cytometry.
The primer that the present invention also provides step (i) to use, its nucleotide sequence is as follows:
The 1st group of primer nucleic acid fragment shown in SEQ ID NO.1 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.7, and its downstream primer nucleotide sequence is shown in SEQ ID NO.8;
The 2nd group of primer nucleic acid fragment shown in SEQ ID NO.2 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.10, and its downstream primer nucleotide sequence is shown in SEQ ID NO.11;
The 3rd group of primer nucleic acid fragment shown in SEQ ID NO.3 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.13, and its downstream primer nucleotide sequence is shown in SEQ ID NO.14;
The 4th group of primer nucleic acid fragment shown in SEQ ID NO.4 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.16, and the downstream primer nucleotide sequence is shown in SEQ ID NO.17;
The 5th group of primer nucleic acid fragment shown in SEQ ID NO.5 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.19, and its downstream primer nucleotide sequence is shown in SEQ ID NO.20;
The 6th group of primer nucleic acid fragment shown in SEQ ID NO.6 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.22, and its downstream primer nucleotide sequence is shown in SEQ ID NO.23.
Corresponding respectively avian influenza virus H5 hypotype, avian influenza virus H7 hypotype, sars coronavirus, Hantaan virus, Ye Shi plague bacillus, the anthrax bacillus of detecting of above-mentioned the 1st group to the 6th group primer.
In actual applications, also has a kind of any one or more than one combination that may be to use in the above-mentioned primer group, increase and detect in influenze virus H 5 subtype, avian influenza virus H7 hypotype, sars coronavirus, Hantaan virus, Ye Shi plague bacillus, the anthrax bacillus a certainly or several, as use the sequence of the 3rd group and the 6th group to carry out detection of sars coronavirus and anthrax bacillus etc.
The capture probe that the present invention also provides described step (ii) to use, its nucleotide sequence are shown in SEQ IDNO.25, and what it was direct or indirect is connected on the solid support, and hybridize with step (i) gained amplified production.
The present invention also provides step the (ii) middle detection probes of using:
Sequence 1 is used to detect the nucleic acid fragment shown in SEQ ID NO.1 shown in SEQ ID NO.9;
Sequence 2 is used to detect the nucleic acid fragment shown in SEQ ID NO.2 shown in SEQ ID NO.12;
Sequence 3 is used to detect the nucleic acid fragment shown in SEQ ID NO.3 shown in SEQ ID NO.15;
Sequence 4 is used to detect the nucleic acid fragment shown in SEQ ID NO.4 shown in SEQ ID NO.18;
Sequence 5 is used to detect the nucleic acid fragment shown in SEQ ID NO.5 shown in SEQ ID NO.21;
Sequence 6 is used to detect the nucleic acid fragment shown in SEQ ID NO.6 shown in SEQ ID NO.24.
Corresponding respectively avian influenza virus H5 hypotype, avian influenza virus H7 hypotype, sars coronavirus, Hantaan virus, Ye Shi plague bacillus, the anthrax bacillus that detects after increasing of above-mentioned sequence 1～6.
Detection probes can detect with multiple biological method.Preferably, described detection probes adds substrate by vitamin H or digoxigenin labeled, carries out result's judgement through the termination reaction step after spectrophotometer reads absorbance.
The present invention preferably embodiment is to carry out the (ii) described detection step of step at one time by a plurality of reaction tubess, and the detection probes that each reaction tubes uses is a kind of and with step (i) the employed primer corresponding common existence that detect a certain pathogenic agent of sequence 1 to the sequence 6.
The absorbancy result treatment of aforesaid method can be taked following scheme: its result treatment can be taked following scheme: test n negative control sample, get its absorbance, determine threshold value: m+K * SD as follows, the negative contrast absorbancy of m arithmetical av, the negative contrast absorbancy of SD standard deviation, K decides according to different experimental conditions, as colony's number or the sample number that detects.Detect reading and then be judged to be detected result " positive " greater than threshold value; Detect reading and then be judged to be detected result " feminine gender " less than threshold value.
Term of the present invention " primer " refers to the single stranded oligonucleotide sequence as the initiation site of synthetic primer extension products, itself and nucleic acid chains complementation to be duplicated, and length and sequence must be suitable for the synthetic of extension products.
Term of the present invention " probe " refers to single stranded oligonucleotide, is used for and the nucleic acid fragment specific hybrid." specific hybrid " refers to that the whole zone of described probe and nucleic acid or a part form duplex under specific experiment condition, and under these conditions, described probe not with detected sample in other nucleic acid of existing or other zones of nucleic acid form duplexs.
After the hybridization of probe and nucleic acid fragment, can be by the mode of any appropriate, the detection of hybridizing, for example label probe that can detect and/or nucleic acid fragment, for auxiliary detection, preferred target nucleic acid fragment carries out NASBA amplification or pcr amplification.
The present invention also provides a kind of test kit that the infectious disease pathogens that may be present in the biological sample are detected, it comprises at least 1 group primer, the described primer nucleic acid fragment of avian influenza virus H5 hypotype HA gene shown in SEQ ID NO.1 that be used to increase, the nucleic acid fragment of avian influenza virus H7 hypotype HA gene shown in SEQ IDNO.2, the nucleic acid fragment of the nucleocapsid protein gene of sars coronavirus shown in SEQ ID NO.3, the nucleic acid fragment of Hantaan virus nucleocapsid protein gene shown in SEQ ID NO.4, nucleic acid fragment or the nucleic acid fragment of anthrax bacillus putative protein gene shown in SEQ ID NO.6 shown in the Ye Shi plague bacillus pPCP1 genes of SEQ ID NO.5.
Preferably, the primer of test kit of the present invention is selected from following 6 groups of primers:
The 1st group of primer nucleic acid fragment shown in SEQ ID NO.1 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.7, and its downstream primer nucleotide sequence is shown in SEQ ID NO.8;
The 2nd group of primer nucleic acid fragment shown in SEQ ID NO.2 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.10, and its downstream primer nucleotide sequence is shown in SEQ ID NO.11;
The 3rd group of primer nucleic acid fragment shown in SEQ ID NO.3 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.13, and its downstream primer nucleotide sequence is shown in SEQ ID NO.14;
The 4th group of primer nucleic acid fragment shown in SEQ ID NO.4 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.16, and the downstream primer nucleotide sequence is shown in SEQ ID NO.17;
The 5th group of primer nucleic acid fragment shown in SEQ ID NO.5 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.19, and its downstream primer nucleotide sequence is shown in SEQ ID NO.20;
The 6th group of primer nucleic acid fragment shown in SEQ ID NO.6 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.22, and its downstream primer nucleotide sequence is shown in SEQ ID NO.23.
Preferably, test kit of the present invention also comprises the capture probe shown in nucleotide sequence SEQ ID NO.25.
More preferably, test kit of the present invention also comprises the following at least a detection probes corresponding with primer:
Sequence 1 is used to detect the nucleic acid fragment shown in SEQ ID NO.1 shown in SEQ ID NO.9;
Sequence 2 is used to detect the nucleic acid fragment shown in SEQ ID NO.2 shown in SEQ ID NO.12;
Sequence 3 is used to detect the nucleic acid fragment shown in SEQ ID NO.3 shown in SEQ ID NO.15;
Sequence 4 is used to detect the nucleic acid fragment shown in SEQ ID NO.4 shown in SEQ ID NO.18;
Sequence 5 is used to detect the nucleic acid fragment shown in SEQ ID NO.5 shown in SEQ ID NO.21;
Sequence 6 is used to detect the nucleic acid fragment shown in SEQ ID NO.6 shown in SEQ ID NO.24.
Test kit of the present invention can also comprise one or more following components:
Hybridization buffer or prepare the specification sheets of described hybridization buffer; Washing soln or prepare the specification sheets of described washing soln; Detect the component of the crossbred that forms; Be used for probe is attached to component on the solid support.
The method that the infectious disease pathogens that may be present in the biological sample are detected of the present invention, the advantage of contrast prior art is:
(1) detection speed is fast, and the efficient height can be as required, and double at random, triple or Multiple Combination detects multiple infective pathogen body simultaneously, also can detect single pathogenic agent;
(2) highly sensitive, the Cmin that NASBA can detect virus is 10
-11PM is higher than the susceptibility of classical pathogen separation method;
(3) high specificity because external double-stranded DNA does not have the T7 promoter sequence, can not be amplified, and this has just significantly improved the specificity of NASBA reaction; Because primer in the amplification procedure and the probe dual function in the testing process, guaranteed the specificity of pathogen detection once more, and the reaction conditions gentleness of NASBA, and shorter than the time of PCR reaction needed, therefore transcribe more faithful to template, further reduced the mispairing rate;
(4) simple to operate, the result is stable, and whole testing process only needs the instrument of this routine of microplate reader;
(5) be applicable to early diagnosis, be applicable to the combine detection at random of the early stage rapid detection of the great communicate illness that FUO causes, multiple pathogenic agent and the check of a large amount of samples.
(6) applied widely, sampling is simple.The pollution of DNA, heparin, EDTA, Citrate trianion, oxyphorase, albumin and lipid etc. will can not impact the result in the sample, thereby is applicable to various samples to be checked such as brush,throat, ight soil, blood etc.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention that those skilled in the art can carry out various transformations and modification to it, and these equivalent form of values fall into protection scope of the present invention equally.
The experimental technique of unreceipted actual conditions among the following embodiment, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
Design and preparation primer, probe sequence.
The specific gene sequence of avian influenza virus H5 hypotype, avian influenza virus H7 hypotype, sars coronavirus, Hantaan virus, Ye Shi plague bacillus, anthrax bacillus is chosen in search according to NCBI, and they are respectively:
Avian influenza virus H5 hypotype HA gene order, the GENEBANK accession number is FM160642, chooses sequence high conservative part as target nucleic acid sequence, and the target sequence that the present invention chooses is HA gene 1457-1653, and its nucleotide sequence is shown in SEQ ID NO.1;
Avian influenza virus H7 hypotype HA gene order, the GENEBANK accession number is AB302789, the target nucleic acid sequence that the present invention chooses be HA because of 452-764, its nucleotide sequence is shown in SEQ ID NO.2;
The nucleocapsid protein gene order of sars coronavirus, GENEBANK accession number are DQ898174, and the target nucleic acid sequence that the present invention chooses is the 18120-18295 of nucleocapsid protein gene, and its nucleotide sequence is shown in SEQ ID NO.3;
Hantaan virus nucleocapsid protein gene order, GENEBANK accession number are NC005218, and the target nucleic acid sequence that the present invention chooses is the 374-624 of nucleocapsid protein gene, and its nucleotide sequence is shown in SEQID NO.4;
Ye Shi plague bacillus pPCP1 gene order, the GENEBANK accession number is M27820, and the target nucleic acid sequence that the present invention chooses is the 560-767 of pPCP1 gene, and its nucleotide sequence is shown in SEQ ID NO.5;
Anthrax bacillus putative protein gene order, GENEBANK accession number are AM404332, and the target nucleic acid sequence that the present invention chooses is the 1346-1536 of putative protein gene, and its nucleotide sequence is shown in SEQ IDNO.6.
Analyze the information of above-mentioned virus-specific gene, carry out between primer/eliminating of interdimers through sequence analysis software DNAman, and through the specificity of BLAST checking primer and with the homology of close pathogenic agent, design NASBA primer and probe at above-mentioned pathogen detection.
The nucleotide sequence of capture probe is shown in SEQ ID NO.25; The detection probes of primer and correspondence is as follows:
The 1st group: the upstream primer nucleotide sequence of the nucleic acid fragment of avian influenza virus H5 hypotype HA gene shown in SEQ ID NO.1 that be used to increase shown in SEQ ID NO.7, the downstream primer nucleotide sequence shown in SEQ ID NO.8, detection probes is shown in SEQ ID NO.9;
The 2nd group: the upstream primer nucleotide sequence of the nucleic acid fragment of avian influenza virus H7 hypotype HA gene shown in SEQ ID NO.2 that be used to increase shown in SEQ ID NO.10, the downstream primer nucleotide sequence shown in SEQ ID NO.11, detection probes is shown in SEQ ID NO.12;
The 3rd group: the upstream primer nucleotide sequence of the nucleic acid fragment of nucleocapsid protein gene shown in SEQ ID NO.3 of the sars coronavirus that is used to increase shown in SEQ ID NO.13, the downstream primer nucleotide sequence shown in SEQ ID NO.14, detection probes is shown in SEQ ID NO.15;
The 4th group: the upstream primer nucleotide sequence of the nucleic acid fragment of Hantaan virus nucleocapsid protein gene shown in SEQ ID NO.4 that be used to increase shown in SEQ ID NO.16, the downstream primer nucleotide sequence shown in SEQ ID NO.17, detection probes is shown in SEQ ID NO.18;
The 5th group: the upstream primer nucleotide sequence of the nucleic acid fragment of Ye Shi plague bacillus pPCP1 gene shown in SEQ ID NO.5 that be used to increase shown in SEQ ID NO.19, the downstream primer nucleotide sequence shown in SEQID NO.20, detection probes is shown in SEQ ID NO.21;
The 6th group: the upstream primer nucleotide sequence of the nucleic acid fragment of anthrax bacillus putative protein gene shown in SEQ ID NO.6 that be used to increase shown in SEQ ID NO.22, the downstream primer nucleotide sequence shown in SEQID NO.23, detection probes is shown in SEQ ID NO.24.
Embodiment 2
Prepare test kit of the present invention.Consist of:
1, amplification system
Every group of 10uM of primer (6 groups)
Tris/HCl 10-100mM
Repone K 1-10mM
BSA 1-5g/ml
Dithiothreitol (DTT) 1-5mM
Nucleoside triphosphate and deoxidation nucleoside triphosphate isoconcentration mixture 200-1000uM
ThermoScript II 5-300U
Ribonuclease H RNase H 5-300U
Phage t7 ribonucleic acid polymerase 5-300U
Negative control is no RNA enzyme water
Positive control is avian influenza virus H5 hypotype HA gene order, avian influenza virus H7 type HA gene, the nucleocapsid protein gene of sars coronavirus, Hantaan virus nucleocapsid protein gene order, Ye Shi plague bacillus pPCP1 gene order, the anthrax bacillus putative protein gene order of 5pM synthetic.
2, detection architecture
Detection probes (6 groups) 26 μ M by digoxigenin labeled
Capture probe (biotin labeling) 26 μ M
Cleaning buffer solution 1x TBS
Hybridization buffer 10mg/ml BSA, 50mM Tris-HCl (pH 7.5)
Detect damping fluid 1x TBS:1.4M NaCl, 30mM KCl, 260mM
Detect the antibody of the anti-digoxin of concentrated solution alkali phosphatase enzyme mark
Stop buffer 3M NaOH
Substrate 10mM para-nitro-pheneye phosphate solution
Negative control is no RNA enzyme water;
Positive control is avian influenza virus H5 hypotype HA gene order, avian influenza virus H7 hypotype HA gene, the nucleocapsid protein gene of sars coronavirus, Hantaan virus nucleocapsid protein gene order, Ye Shi plague bacillus pPCP1 gene order, the anthrax bacillus putative protein gene order of 5pM synthetic.
Embodiment 3:
To the method that the infectious disease pathogens that may be present in the biological sample detect, also be the using method of test kit of the present invention simultaneously.
1, nucleic acid extraction
Get sputum sample to be measured, the centrifuging and taking supernatant, add the 1ml guanidinium isothiocyanate, add 1ml TRIZOL behind the mixing again, the vibration mixing was placed 5 minutes in the ice bath. and add the 350ul chloroform, mixing fully vibrates, after the static layering, immediately in 4 ℃, centrifugal 20 minutes of 12000r/min. supernatant is transferred to another centrifuge tube, adds isopyknic Virahol (4 ℃ of precoolings) and place 1h for mixing .-20 ℃, 4 ℃, 12000r/ minute centrifugal 20 minutes, precipitation was that total RNA. adds 0.5ml 75% washing with alcohol, 4 ℃, 12000r/ minute centrifugal 10 minutes, carefully outwell ethanol, room temperature was placed 10 minutes, added an amount of DEPC treated water dissolution precipitation, promptly obtain the determined nucleic acid sample ,-80 ℃ of preservations are standby.
2, NASBA amplified reaction
20 μ l NASBA amplification reaction systems shown in being formulated as follows in 0.5ml does not have the sterilization centrifuge tube of RNA enzyme, 41 ℃ of incubations 90 minutes, nucleic acid amplification product was used for subsequent detection.
The final concentration of NASBA amplification reaction system (20 μ l) is specially:
Determined nucleic acid sample 200 μ M
Primer 1 μ M
AMV 10U
RNase?H 10U
T7 rna polymerase 10U
Tris/HCl 10mM
Repone K 1mM
BSA 100mg/ml
Dithiothreitol (DTT) 1mM
Nucleoside triphosphate and deoxidation nucleoside triphosphate isoconcentration mixture 200uM
3, the NASBA amplified production detects
Get step 2 gained 5 μ l NASBA products, add 1 μ l detection probes solution (every hole adds a kind of) and 1 μ l capture probe solution, with add behind the 43 μ l hybridization buffer mixings bag by the microwell plate of streptavidin (available from Thermo fisher scientific) each independently in the hole, 45 ℃ of incubations 1 hour, with 250 μ l 1x TBS, pH 7.4 cleans aperture three times.Incline at every turn and will pat dry microwell plate behind the solution, every hole adds 100 μ l and detects damping fluid and the mixture that detects concentrated solution (by detecting concentrated solution: detect damping fluid=dilution in 1: 500), detect concentrated solution available from Sigma, P/N N7653, at room temperature incubation is 30 minutes, with 250 μ l 1x TBS, pH 7.4 cleans aperture three times, incline at every turn and will pat dry microwell plate behind the solution, micropore adds 100 μ l substrates, lucifuge room temperature incubation 5 minutes, 100 μ l stop buffers also shake the color development stopping reaction gently, and microwell plate and microwell plate sheet frame are put into standard 96 hole microwell plate spectrophotometers and read the 405nm absorbancy.100 μ l substrates add 100 μ l stop buffer references as a setting.
4, detected result analysis
Above-mentioned absorbancy result treatment is taked following scheme: test 8 its absorbances of negative control sample determination, determine threshold value: m+8 * SD, the negative contrast absorbancy of m arithmetical av, the negative contrast absorbancy of SD standard deviation as follows.Threshold value is 0.15+0.035 * 8=0.43.Detect reading and then be judged to be " positive " greater than threshold value; Detect reading and then be judged to be " feminine gender " less than threshold value.
Embodiment 4:
To the method that the infectious disease pathogens that may be present in the biological sample detect, also be the using method of test kit of the present invention simultaneously.
1, nucleic acid extraction
Get blood sample to be measured, get fresh whole blood 2ml and add 1ml 3% Trisodium Citrate, behind the mixing 4 ℃, 3000r/ minute centrifugal, 10 minutes, get supernatant, add the 1ml guanidinium isothiocyanate, add 1ml TRIZOL behind the mixing again, the vibration mixing was placed 5 minutes in the ice bath. and add the 350ul chloroform, mixing fully vibrates, after the static layering, in 4 ℃, 12000r/ minute is centrifugal 20 minutes immediately. and supernatant is transferred to another centrifuge tube, adds isopyknic Virahol (4 ℃ of precoolings) and place 1h for mixing .-20 ℃, 4 ℃, centrifugal 20 minutes of 12000r/min, precipitation is that total RNA. adds 0.5ml 75% washing with alcohol, 4 ℃, centrifugal 10 minutes of 12000r/min, carefully outwell ethanol, room temperature was placed 10 minutes, added an amount of DEPC treated water dissolution precipitation, promptly obtain the determined nucleic acid sample ,-80 ℃ of preservations are standby.
2, NASBA amplification
20 μ l NASBA amplification reaction systems shown in being formulated as follows in 0.5ml does not have the sterilization centrifuge tube of RNA enzyme, 41 ℃ of incubations 90 minutes, nucleic acid amplification product was used for subsequent detection.
The final concentration of NASBA amplification reaction system (20 μ l) is specially:
Determined nucleic acid sample 600uM
Primer 2 uM
AMV 150U
RNase?H 150U
T7 rna polymerase 150U
Tris/HCl 10mM
Repone K 1mM
BSA 200mg/ml
Dithiothreitol (DTT) 3mM
Nucleoside triphosphate and deoxidation nucleoside triphosphate isoconcentration mixture 600uM
3, the NASBA amplified production detects
Get step 2 gained 5 μ l NASBA products, add 1 μ l detection probes solution (every hole adds a kind of) and 1 μ l capture probe solution, with 43 μ l hybridization buffer mixings and add bag by the microwell plate of streptavidin (available from Thermo fisher scientific company) each independently in the hole, 45 ℃ of incubations 1 hour, with 250 μ l 1x TBS, pH 7.4 cleans aperture three times.Incline at every turn and will pat dry microwell plate behind the solution, every hole adds the mixture (by detecting concentrated solution: detect damping fluid=configuration in 1: 500) that 100 μ l detect damping fluid and detect damping fluid, at room temperature incubation is 30 minutes, with 250 μ l 1x TBS, pH 7.4 cleans aperture three times, incline at every turn and will pat dry microwell plate behind the solution, micropore adds 100 μ l 10mM Tris/HCl, lucifuge room temperature incubation 5 minutes, add 100 μ l stop buffers and shake the color development stopping reaction gently, microwell plate and microwell plate sheet frame are put into standard 96 hole microwell plate spectrophotometers and read the 405nm absorbancy, use 100 μ l 10mM Tris/HCl to add 100 μ l stop buffer references as a setting.
When whether containing certain 3 kinds of pathogenic agent in detecting testing sample, only need to add the primer of these 3 kinds of pathogenic agent correspondences respectively at 3 reaction tubess when the preparation amplification system, add the probe solution of these 3 kinds of pathogenic agent correspondences during detection, other steps together.
4, detected result analysis
Measure the absorbancy of 10 known negative samples, threshold value is m (negative control absorbancy arithmetical av)+10 * SD (negative control absorbancy standard deviation), and threshold value is generally 0.15+0.03 * 10=0.45.Detect reading and then be judged to be detected result " positive " greater than threshold value; Detect reading and then be judged to be detected result " feminine gender " less than threshold value.
Embodiment 5: the sensitivity experiment that detects the H7 subtype avian influenza virus
1, nucleic acid amplification
Get 1ml H7 subtype avian influenza virus standard substance and add the 1ml guanidinium isothiocyanate, add 1ml TRIZOL behind the mixing again, the vibration mixing, placed 5 minutes in the ice bath. add the 350ul chloroform, mixing fully vibrates, after the static layering, immediately in 4 ℃, centrifugal 20 minutes of 12000r/min. supernatant is transferred to another centrifuge tube, adds isopyknic Virahol (4 ℃ of precoolings) and place 1h for mixing .-20 ℃, 4 ℃, centrifugal 20 minutes of 12000r/min, precipitation is that total RNA. adds 0.5ml 75% washing with alcohol, 4 ℃, centrifugal 10 minutes of 12000r/min, carefully outwell ethanol, room temperature was placed 10 minutes, added an amount of DEPC treated water dissolution precipitation, promptly obtain the determined nucleic acid sample, measure its concentration.
There is not in the sterilization centrifuge tube of RNA enzyme mark respectively at 0.5ml, 20 μ lNASBA amplification reaction systems shown in being formulated as follows, and two negative controls are set simultaneously, H7 subtype avian influenza virus RNA is formulated as 10
-9Pm, 10
-10Pm, 10
-11Pm, 10
-12The final concentration of pm, every kind of concentration is provided with multiple pipe, with no RNA enzyme water as negative control, 41 ℃ of water-baths 95 minutes.Nucleic acid amplification product is used for subsequent detection.
The final concentration of NASBA amplification reaction system (20 μ l) is specially:
H7 type avian influenza virus RNA
Primer (the 2nd group) 1 μ M
AMV 10U
RNase?H 10U
T7 rna polymerase 10U
Tris/HCl 10mM
Repone K 1mM
BSA 100mg/ml
Dithiothreitol (DTT) 1mM
Nucleoside triphosphate and deoxidation nucleoside triphosphate isoconcentration mixture 200uM
Get amplified production and entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to carry out sequential analysis, its nucleotide sequence is shown in SEQ ID NO.2, and is consistent with avian influenza virus H7 hypotype HA gene order 452-764 section.
2, detection of nucleic acids
1) supporting to lay a microwell plate lath on the sheet frame, this coated in microporous plate streptavidin (available from Thermo fisher scientific company).
2) add successively in each micropore 43 μ l hybridization buffers, add 1 μ l detection probes solution (group 2) and 1 μ l capture probe solution, 5 μ l amplified productions rap supporting plate with mixing solutions (attention avoids producing bubble).
3) firmly cover and seal film and avoid evaporation, 45 ℃ of water-baths 60 minutes.
4) incline behind the solution, with 250 μ l 1x TBS, pH 7.4 cleans apertures five times, and cleans 30s at every turn, pats dry microwell plate behind the solution that inclines.
5) every hole adds mixture that 100 μ l detect damping fluid and detect concentrated solution (by detecting concentrated solution: damping fluid=configuration in 1: 500), firmly cover and seal film and avoid evaporation.
6) incubation 30 minutes at room temperature.
7) incline behind the solution, with 250 μ l 1x TBS, pH 7.4 cleans apertures five times, and cleans 30s at every turn, pats dry microwell plate behind the solution that at every turn inclines.
8) micropore adds 100 μ l and connects liquid (carefully avoiding introducing foam in the hole).
9) lucifuge room temperature incubation is 5 minutes.
10) micropore adds 100 μ l stop buffers and shakes the color development stopping reaction gently.
11) microwell plate and microwell plate sheet frame are put into standard 96 hole microwell plate spectrophotometers and read the 405nm absorbancy.
3, the result is as shown in table 1.
Table 1. the method for the invention detects the absorbance of different concns H7 subtype avian influenza virus
Do not have
The inferior H7 H7 of H7 H7 H7 H7 H7 H7
Sample RNA RNA
Hypotype hypotype hypotype hypotype type hypotype hypotype hypotype
Enzyme water enzyme water concentration
10
-9 10
-9 10
-10 10
-10 10
-11 10
-11 10
-12 10
-12
(pM)
The OD value
1.5 1.443?1.203 1.155 0.579 0.635 0.32 0.341 0.114 0.189
405nm
4, interpretation of result
Read absorbance through spectrophotometer and carry out result's judgement, measure the absorbancy of 7 known negative samples (water), threshold value is m (negative control absorbancy arithmetical av)+7 * SD (negative control absorbancy standard deviation), and threshold value is 0.17+0.04 * 7=0.45.Detect reading and then be judged to be detected result " positive " greater than threshold value; Detect reading and then be judged to be detected result " feminine gender " less than threshold value.Be that the minimum concentration that the detection probes of the primer of group 2 and group 2 can detect the H7 subtype avian influenza virus is 10
-11PM.
Embodiment 6: the sensitivity experiment that detects other pathogenic agent
With reference to embodiment 5, avian influenza virus H5 hypotype, sars coronavirus, Hantaan virus, Ye Shi plague bacillus or the anthrax bacillus of preparation different concns carry out NASBA amplification back with its corresponding primer and measure the amplified production sequence, and its Nucleotide is consistent with target gene.Detect with corresponding capture probe and detection probes, detected minimum concentration all can reach 10
-11PM.
Embodiment 7: detect the primer specificity test of H7 subtype avian influenza virus
The 468-471 position of the gene fragment of avian influenza virus H7 hypotype HA gene shown in SEQ ID NO.2 is suddenlyd change, nucleotide sequence after the sudden change is shown in SEQ ID NO.26, in order to narrate conveniently, be referred to as the interference sequence of bird flu H7 hypotype HA gene fragment.
Get isocyatic embodiment 5 amplification gained H7 subtype avian influenza virus, above-mentioned interference sequence and people DNA and mix, be mixed with testing sample.There is not in the sterilization centrifuge tube of RNA enzyme mark respectively at 0.5ml, 20 μ l NASBA amplification reaction systems shown in being formulated as follows, and two negative controls are set simultaneously, 41 ℃ of water-baths 95 minutes.Nucleic acid amplification product is used for subsequent detection.
The final concentration of NASBA amplification reaction system (20 μ l) is specially:
Testing sample 800 μ M
Primer (the 2nd group) 1 μ M
AMV 10U
RNase?H 10U
T7 RNA polymerase 10U
Tris/HCl 10mM
Repone K 1mM
BSA 100mg/ml
Dithiothreitol (DTT) 1mM
Nucleoside triphosphate and deoxidation nucleoside triphosphate isoconcentration mixture 200uM
Get amplified production and entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to carry out sequential analysis, its sequence is one section of 5 of nucleotide sequence shown in SEQ ID NO.2 ' connected and capture probe complementary sequence.
Embodiment 8: the specificity experiment of other primers
With reference to embodiment 7, with the nucleic acid fragment of avian influenza virus H5 hypotype HA gene shown in SEQ ID NO.1, the nucleic acid fragment of the nucleocapsid protein gene of sars coronavirus shown in SEQ ID NO.3, the nucleic acid fragment of Hantaan virus nucleocapsid protein gene shown in SEQ ID NO.4, the interference sequence and the target nucleic acid fragment that form behind nucleic acid fragment shown in the Ye Shi plague bacillus pPCP1 genes of SEQ ID NO.5 and the several base random mutations of the nucleic acid fragment of anthrax bacillus putative protein gene shown in SEQID NO.6, after people DNA isoconcentration mixes, increase with corresponding primer, get amplified production and entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to carry out sequential analysis, its sequence is one section of target nucleic acid sequence 5 ' connected and capture probe complementary sequence.
Above experimental result illustrates that primer specificity provided by the invention is strong, analyzes reason and is not have the T7 promoter sequence owing to external double-stranded DNA, can not be amplified, and this has just significantly improved the specificity of NASBA reaction; And the reaction conditions gentleness of NASBA, and shorter than the time of PCR reaction needed, therefore transcribe more faithful to template, further reduced the mispairing rate.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
SEQUENCE?LISTING
＜110〉Beijing Haikang DNA Chips Co., Ltd.
＜120〉a kind of method and test kit that detects infectious disease pathogens
<130>MP090690
<160>26
<170>PatentIn?version?3.3
<210>1
<211>197
<212>DNA
＜213〉avian influenza virus H5 hypotype HA gene 1457-1653
<400>1
gtgacaatga?atgcatggaa?agtgtgagaa?atggaacgta?tgactatcca?caatactcag 60
aagaatcaag?gctaaacagg?gaggaaatag?atggagtcaa?attggaatca?atgggcactt 120
atcagatact?atcaatctac?tcaacagtgg?cgagttccct?agcactggca?atcatggtag 180
ctggtctgtc?tttttgg 197
<210>2
<211>313
<212>DNA
＜213〉452-764 of avian influenza virus H7 hypotype HA gene
<400>2
agatcaggat?cttcattcta?tgcagagatg?aaatggcttc?tgtcgaatac?agacaatgct 60
gctttcccgc?aaatgacgaa?atcgtacaaa?aacacaagga?aggatccagc?tctgatagtc 120
tgggggattc?atcattccgg?atcaaccaca?gaacagacca?aattatatgg?aagtgggaac 180
aagttaataa?cagttgggag?ctccaattat?caacagtcct?ttgtaccaag?tccgggggcg 240
agaccacaag?tgaatggcca?atctggacgg?atcgatttcc?attggctaat?actgaattcc 300
aatgacacag?tca 313
<210>3
<211>176
<212>DNA
＜213〉18120-18295 of sars coronavirus nucleocapsid protein gene
<400>3
taccgtagac?tcatctctat?gatgggtttc?aaaatgaatt?accaagtcaa?tggttaccct 60
aatatgttta?tcacccgcga?agaagctatt?cgtcacgttc?gtgcgtggat?tggctttgat 120
gtagagggct?gtcatgcaac?tagagatgct?gtgggtacta?acctacctct?ccagct 176
<210>4
<211>251
<212>DNA
＜213〉374-624 of Hantaan virus nucleocapsid protein gene
<400>4
caggacagac?agcagactgg?ctgagcatca?tcgtctatct?tacatccttt?gtcgtcccga 60
tacttctgaa?agctctgtat?atgttgacaa?caagggggag?gcaaactacc?aaggataata 120
aagggacccg?gattcgattt?aaggatgata?gctcgttcga?ggatgttaac?ggtatccgga 180
aaccaaaaca?tctttacgtg?tccttgccaa?atgcacagtc?aagcatgaag?gcagaagaga 240
ttacacctgg?t 251
<210>5
<211>208
<212>DNA
＜213〉560-767 of Ye Shi plague bacillus pPCP1 gene
<400>5
tggcttccgg?gtcaggtaat?atggatgact?acgactggat?gaatgaaaat?caatctgagt 60
ggacagatca?ctcatctcat?cctgctacaa?atgttaatca?tgccaatgaa?tatgacctca 120
atgtgaaagg?ctggttactc?cagaatgaga?attataaagc?aggtataaca?gcaggatatc 180
aggaaacacg?tttcagttgg?acagctac 208
<210>6
<211>191
<212>DNA
＜213〉1346-1536 of anthrax bacillus putative protein gene
<400>6
aagattagat?acggatcaag?tatatgggaa?tatagcaaca?tacaattttg?aaaatggaag 60
agtgagggtg?gatacaggct?cgaactggag?tgaagtgtta?ccgcaaattc?aagaaacaac 120
tgcacgtatc?atttttaatg?gaaaagattt?aaatctggta?gaaaggcgga?tagcggcggt 180
taatcctagt?g 191
<210>7
<211>40
<212>DNA
＜213〉primer H5-F
<400>7
gatgcaaggt?cgcatatgag?gtgacaatga?atgcatggaa 40
<210>8
<211>51
<212>DNA
＜213〉primer H5-R
<400>8
aattctaata?cgactcacta?tagggagaag?gccataaaga?cagaccagcc?a 51
<210>9
<211>23
<212>DNA
＜213〉probe H5-P
<400>9
gcaagttccc?tagcactggc?aat 23
<210>10
<211>52
<212>DNA
＜213〉primer H7-F
<400>10
gatgcaaggt?cgcatatgag?agaacaggat?cttcattcta?tgcagagatg?aa 52
<210>11
<211>46
<212>DNA
＜213〉primer H7-R
<400>11
aattctaata?cgactcacta?tagggagaag?gtaactgtgt?cattag 46
<210>12
<211>21
<212>DNA
＜213〉probe H7-P
<400>12
gaccacaagt?aaatggacaa?t 21
<210>13
<211>42
<212>DNA
＜213〉primer SARS-F
<400>13
gatgcaaggt?cgcatatgag?taccgtagac?tcatctctat?ga 42
<210>14
<211>56
<212>DNA
＜213〉primer SARS-R
<400>14
aattctaata?cgactcacta?tagggagaag?gagctggaga?ggtaggttag?taccca 56
<210>15
<211>21
<212>DNA
＜213〉probe SARS-P
<400>15
gtttatcacc?cgcgaagaag?c 21
<210>16
<211>38
<212>DNA
＜213〉primer Hantaan-F
<400>16
gatgcaaggt?cgcatatgag?cagggcaaac?agcagact 38
<210>17
<211>52
<212>DNA
＜213〉primer Hantaan-R
<400>17
aattctaata?cgactcacta?tagggagaag?gaccaggtgt?aagctcatca?gc 52
<210>18
<211>24
<212>DNA
＜213〉probe Hantaan-P
<400>18
agcatcatcgtctatcttac?atcc 24
<210>19
<211>42
<212>DNA
＜213〉primer Yersinia-F
<400>19
gatgcaaggt?cgcatatgag?tggcttccgg?gtcaggtaat?at 42
<210>20
<211>55
<212>DNA
＜213〉primer Yersinia-R
<400>20
aattctaata?cgactcacta?tagggagaag?ggtagctgtc?caactgaaac?gtgtt 55
<210>21
<211>24
<212>DNA
＜213〉probe Yersinia-P
<400>21
tatgacctca?atgtgaaagg?ctgg 24
<210>22
<211>48
<212>DNA
＜213〉primer Bacillus-F
<400>22
gatgcaaggt?cgcatatgag?aagattagat?acggatcaag?tatatggg 48
<210>23
<211>53
<212>DNA
＜213〉primer Bacillus-R
<400>23
aattctaata?cgactcacta?tagggagaag?gcactaggat?taaccgccgc?tat 53
<210>24
<211>24
<212>DNA
＜213〉probe Bacillus-P
<400>24
tggagtgaag?tgttaccgca?aatt 24
<210>25
<211>20
<212>DNA
＜213〉capture probe
<400>25
gatgcaaggt?cgcatatgag 20
<210>26
<211>313
<212>DNA
＜213〉interference sequence of bird flu H7 hypotype HA gene fragment
<400>26
agatcaggat?cttcatctct?tgcagagatg?aaatggcttc?tgtcgaatac?agacaatgct 60
gctttcccgc?aaatgacgaa?atcgtacaaa?aacacaagga?aggatccagc?tctgatagtc 120
tgggggattc?atcattccgg?atcaaccaca?gaacagacca?aattatatgg?aagtgggaac 180
aagttaataa?cagttgggag?ctccaattat?caacagtcct?ttgtaccaag?tccgggggcg 240
agaccacaag?tgaatggcca?atctggacgg?atcgatttcc?attggctaat?actgaattcc 300
aatgacacag?tca 313
Claims (10)
1, a kind of method that the infectious disease pathogens that may be present in the biological sample are detected, described infectious disease pathogens comprise avian influenza virus H5 hypotype, avian influenza virus H7 hypotype, sars coronavirus, Hantaan virus, Ye Shi plague bacillus and anthrax bacillus, comprising:
(i) nucleic acid fragment of amplification biological sample, described nucleic acid fragment is the combination of following one or more:
The nucleic acid fragment of avian influenza virus H5 hypotype HA gene shown in SEQ ID NO.1;
The nucleic acid fragment of avian influenza virus H7 hypotype HA gene shown in SEQ ID NO.2;
The nucleic acid fragment of the nucleocapsid protein gene of sars coronavirus shown in SEQ ID NO.3;
The nucleic acid fragment of Hantaan virus nucleocapsid protein gene shown in SEQ ID NO.4;
The nucleic acid fragment of Ye Shi plague bacillus pPCP1 gene shown in SEQ ID NO.5;
The nucleic acid fragment of anthrax bacillus putative protein gene shown in SEQ ID NO.6;
(ii) detect a kind of specific hybrid in described probe and step (i) amplified production with probe.
2, the method for claim 1 is characterized in that, described step (i) is utilized the nucleic acid fragment of NASBA technology amplification sample, and the NASBA operational conditions is 41 ℃～45 ℃ incubations 90～150 minutes.
3, method according to claim 1 and 2 is characterized in that, step (ii) described in the hybridization of probe and step (i) gained amplified production on solid support, carry out.
4, the method for claim 1 is characterized in that, the combination of one or more of 6 groups of oligonucleotide sequences below described step (i) is used is increased as primer:
The 1st group of primer nucleic acid fragment shown in SEQ ID NO.1 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.7, and its downstream primer nucleotide sequence is shown in SEQ ID NO.8;
The 2nd group of primer nucleic acid fragment shown in SEQ ID NO.2 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.10, and its downstream primer nucleotide sequence is shown in SEQ ID NO.11;
The 3rd group of primer nucleic acid fragment shown in SEQ ID NO.3 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.13, and its downstream primer nucleotide sequence is shown in SEQ ID NO.14;
The 4th group of primer nucleic acid fragment shown in SEQ ID NO.4 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.16, and the downstream primer nucleotide sequence is shown in SEQ ID NO.17;
The 5th group of primer nucleic acid fragment shown in SEQ ID NO.5 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.19, and its downstream primer nucleotide sequence is shown in SEQ ID NO.20;
The 6th group of primer nucleic acid fragment shown in SEQ ID NO.6 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.22, and its downstream primer nucleotide sequence is shown in SEQ ID NO.23.
5, method according to claim 4 is characterized in that, described step is (ii) used capture probe, and its nucleotide sequence is shown in SEQ ID NO.25, and what described capture probe was direct or indirect is connected on the solid support.
6, method according to claim 1 or 5 is characterized in that, step uses in (ii) following nucleotide sequence as detection probes:
Sequence 1 is used to detect the nucleic acid fragment shown in SEQ ID NO.1 shown in SEQ ID NO.9;
Sequence 2 is used to detect the nucleic acid fragment shown in SEQ ID NO.2 shown in SEQ ID NO.12;
Sequence 3 is used to detect the nucleic acid fragment shown in SEQ ID NO.3 shown in SEQ ID NO.15;
Sequence 4 is used to detect the nucleic acid fragment shown in SEQ ID NO.4 shown in SEQ ID NO.18;
Sequence 5 is used to detect the nucleic acid fragment shown in SEQ ID NO.5 shown in SEQ ID NO.21;
Sequence 6 is used to detect the nucleic acid fragment shown in SEQ ID NO.6 shown in SEQ ID NO.24.
7, a kind of test kit that the infectious disease pathogens that may be present in the biological sample are detected, it comprises at least 1 group primer, described primer be used to increase nucleic acid fragment, the nucleic acid fragment shown in SEQ ID NO.2, the nucleic acid fragment shown in SEQ ID NO.3, the nucleic acid fragment shown in SEQ IDNO.4, the nucleic acid fragment shown in SEQ ID NO.5 or the nucleic acid fragment shown in SEQ ID NO.6 shown in SEQ ID NO.1.
8, test kit as claimed in claim 7, described primer is selected from:
The 1st group of primer nucleic acid fragment shown in SEQ ID NO.1 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.7, and its downstream primer nucleotide sequence is shown in SEQ ID NO.8;
The 2nd group of primer nucleic acid fragment shown in SEQ ID NO.2 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.10, and its downstream primer nucleotide sequence is shown in SEQ ID NO.11;
The 3rd group of primer nucleic acid fragment shown in SEQ ID NO.3 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.13, and its downstream primer nucleotide sequence is shown in SEQ ID NO.14;
The 4th group of primer nucleic acid fragment shown in SEQ ID NO.4 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.16, and the downstream primer nucleotide sequence is shown in SEQ ID NO.17;
The 5th group of primer nucleic acid fragment shown in SEQ ID NO.5 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.19, and its downstream primer nucleotide sequence is shown in SEQ ID NO.20;
The 6th group of primer nucleic acid fragment shown in SEQ ID NO.6 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.22, and its downstream primer nucleotide sequence is shown in SEQ ID NO.23.
9, test kit according to claim 8 is characterized in that, also comprises the capture probe shown in nucleotide sequence SEQ ID NO.25.
10, according to each described test kit of claim 7 to 9, it is characterized in that, also comprise the following at least a detection probes corresponding with primer:
Sequence 1 is used to detect the nucleic acid fragment shown in SEQ ID NO.1 shown in SEQ ID NO.9;
Sequence 2 is used to detect the nucleic acid fragment shown in SEQ ID NO.2 shown in SEQ ID NO.12;
Sequence 3 is used to detect the nucleic acid fragment shown in SEQ ID NO.3 shown in SEQ ID NO.15;
Sequence 4 is used to detect the nucleic acid fragment shown in SEQ ID NO.4 shown in SEQ ID NO.18;
Sequence 5 is used to detect the nucleic acid fragment shown in SEQ ID NO.5 shown in SEQ ID NO.21;
Sequence 6 is used to detect the nucleic acid fragment shown in SEQ ID NO.6 shown in SEQ ID NO.24.
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| CN102382907A (en) * | 2011-11-10 | 2012-03-21 | 中华人民共和国大榭出入境检验检疫局 | Degenerate reverse transcription-polymerase chain reaction (RT-PCR) detection reagent and kit for hantavirus group |
| CN102382906A (en) * | 2011-11-10 | 2012-03-21 | 中华人民共和国大榭出入境检验检疫局 | Degenerate RT-PCR detection method of Hantavirus group |
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| CN105039597A (en) * | 2015-08-03 | 2015-11-11 | 博奥生物集团有限公司 | Kit used for detecting influenza viruses and application thereof |
| CN105112559A (en) * | 2015-08-03 | 2015-12-02 | 博奥生物集团有限公司 | Kit for detecting coronavirus and application of kit |
| CN105039597B (en) * | 2015-08-03 | 2017-11-10 | 博奥生物集团有限公司 | A kind of kit and its application for being used to detect influenza virus |
| CN105112559B (en) * | 2015-08-03 | 2017-12-08 | 博奥生物集团有限公司 | A kind of kit and its application for being used to detect coronavirus |
| CN113249516A (en) * | 2020-02-10 | 2021-08-13 | 华南理工大学 | Primer group of 2019-nCoV and 2019-nCoV nucleic acid detection kit |
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