CN101545015A

CN101545015A - Method for detecting infectious disease pathogens and kit

Info

Publication number: CN101545015A
Application number: CN200910083898A
Authority: CN
Inventors: 于常海; 刘乐庭; 冯晓燕
Original assignee: BEIJING HAIKANG DNA CHIPS Co Ltd
Current assignee: Hai Kang Life Corp Ltd
Priority date: 2009-05-11
Filing date: 2009-05-11
Publication date: 2009-09-30

Abstract

The invention discloses a method for detecting infectious disease pathogens possibly existing in a biological sample. The infectious disease pathogens comprise influenza A subtype virus, influenza B subtype virus, human parainfluenza 1 subtype virus, human parainfluenza 2 subtype virus, human parainfluenza 3 subtype virus, human parainfluenza 4 subtype virus, respiratory syncytial virus, rubella virus and coxsackie virus. The method comprises the following steps: expanding nucleic acid fragments of the biological sample, and detecting the nucleic acid fragments by using a probe. The invention also provides primers used for expanding and the probe used for detection. The invention also provides a kit comprising the primers. The method has the advantages of high sensitivity, strong specificity, simple operation and wide sample range, can synchronously detect various infectious disease pathogens, and is suitable for early diagnosis of respiratory infectious diseases.

Description

A kind of method and test kit that detects infectious disease pathogens

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 to 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, but must prepare at detecting behind the antibody of this pathogenic agent, especially under the situation that does not also have antibody sources, not be suitable for the early diagnosis of transmissible disease.

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 T7 RNA 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.

Influenza virus A, B hypotype, human parainfluenza virus 1, the respiratory infectious disease common symptoms that 2,3,4 hypotypes, respiratory syncytial virus, rubella virus, Coxsackie virus cause is fever, runny nose and other influenza-like symptom, main by respiratory infectious, morbidity is anxious, infection is fast.These pathogenic agent all are RNA viruses, pathogenic height 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 nine 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 be provided, 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 shown in SEQ ID NO.1 of influenza virus A hypotype stromatin 1 gene;

The nucleic acid fragment shown in SEQ ID NO.2 of the 8th sections gene of influenza virus B hypotype;

The nucleic acid fragment shown in SEQ ID NO.3 of human parainfluenza virus's 1 hypotype nucleocapsid protein gene;

The nucleic acid fragment shown in SEQ ID NO.4 of human parainfluenza virus's 2 hypotype nucleocapsid protein genes;

The nucleic acid fragment shown in SEQ ID NO.5 of human parainfluenza virus's 3 hypotype nucleocapsid protein genes;

The nucleic acid fragment shown in SEQ ID NO.6 of human parainfluenza virus's 4 hypotype phosphoric acid protein genes;

The nucleic acid fragment shown in SEQ ID NO.7 of respiratory syncytial virus N gene;

The nucleic acid fragment shown in SEQ ID NO.8 of glycoprotein E 1 gene of rubella virus;

The genomic nucleic acid fragment shown in SEQ ID NO.9 of Coxsackie B virus 1 type;

(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.10, and its downstream primer nucleotide sequence is shown in SEQ ID NO.11;

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.13, and its downstream primer nucleotide sequence is shown in SEQ ID NO.14;

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.16, and its downstream primer nucleotide sequence is shown in SEQ ID NO.17;

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.19, and the downstream primer nucleotide sequence is shown in SEQ ID NO.20;

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.22, and its downstream primer nucleotide sequence is shown in SEQ ID NO.23;

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.25, and its downstream primer nucleotide sequence is shown in SEQ ID NO.26;

The 7th group of primer nucleic acid fragment shown in SEQ ID NO.7 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.28, and its downstream primer nucleotide sequence is shown in SEQ ID NO.29;

The 8th group of primer nucleic acid fragment shown in SEQ ID NO.8 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.31, and its downstream primer nucleotide sequence is shown in SEQ ID NO.32;

The 9th group of primer nucleic acid fragment shown in SEQ ID NO.9 that is used to increase, its upstream primer nucleotide sequence is shown in SEQ ID NO.34, and its downstream primer nucleotide sequence is shown in SEQ ID NO.35.

Corresponding respectively influenza virus A hypotype, influenza virus B hypotype, human parainfluenza virus's 1 hypotype, human parainfluenza virus's 2 hypotypes, human parainfluenza virus's 3 hypotypes, human parainfluenza virus's 4 hypotypes, respiratory syncytial virus, rubella virus, the Coxsackie virus of detecting of above-mentioned the 1st group to the 9th 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 influenza virus A hypotype, influenza virus B hypotype, human parainfluenza virus's 1 hypotype, human parainfluenza virus's 2 hypotypes, human parainfluenza virus's 3 hypotypes, human parainfluenza virus's 4 hypotypes, respiratory syncytial virus, rubella virus and the Coxsackie virus a certainly or several, as use the sequence of the 3rd group and the 7th group to carry out detection of human parainfluenza virus's 1 hypotype and respiratory syncytial virus etc.

The capture probe that the present invention also provides described step (ii) to use, its nucleotide sequence are shown in SEQ IDNO.37, 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 detects the nucleic acid fragment shown in SEQ ID NO.1 shown in SEQ ID NO.12;

Sequence 2 detects the nucleic acid fragment shown in SEQ ID NO.2 shown in SEQ ID NO.15;

Sequence 3 detects the nucleic acid fragment shown in SEQ ID NO.3 shown in SEQ ID NO.18;

Sequence 4 detects the nucleic acid fragment shown in SEQ ID NO.4 shown in SEQ ID NO.21;

Sequence 5 detects the nucleic acid fragment shown in SEQ ID NO.5 shown in SEQ ID NO.24;

Sequence 6 detects the nucleic acid fragment shown in SEQ ID NO.6 shown in SEQ ID NO.27;

Sequence 7 detects the nucleic acid fragment shown in SEQ ID NO.7 shown in SEQ ID NO.30;

Sequence 8 detects the nucleic acid fragment shown in SEQ ID NO.8 shown in SEQ ID NO.33;

Sequence 9 detects the nucleic acid fragment shown in SEQ ID NO.9 shown in SEQ ID NO.36.

Corresponding respectively influenza virus A hypotype, influenza virus B hypotype, human parainfluenza virus's 1 hypotype, human parainfluenza virus's 2 hypotypes, human parainfluenza virus's 3 hypotypes, human parainfluenza virus's 4 hypotypes, respiratory syncytial virus, rubella virus and the Coxsackie virus 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 9.

The absorbancy result treatment of aforesaid method can be taked following scheme: a tested K 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, and it comprises the primer of influenza virus A hypotype stromatin 1 nucleic acid fragment shown in SEQ ID NO.1 that is used to increase, the primer of the 8th sections gene nucleic acid fragment shown in SEQ ID NO.2 of influenza virus B hypotype is used to increase, the primer of human parainfluenza virus's 1 hypotype nucleocapsid protein gene nucleic acid fragment shown in SEQ IDNO.3 is used to increase, the primer of human parainfluenza virus's 2 hypotype nucleocapsid protein genes nucleic acid fragment shown in SEQ ID NO.4 is used to increase, the primer of human parainfluenza virus's 3 hypotype nucleocapsid protein genes nucleic acid fragment shown in SEQ ID NO.5 is used to increase, the primer of human parainfluenza virus's 4 hypotype phosphoric acid protein genes nucleic acid fragment shown in SEQ ID NO.6 is used to increase, the primer of respiratory syncytial virus N gene nucleic acid fragment shown in SEQ ID NO.7 is used to increase, the primer of glycoprotein E 1 gene nucleic acid fragment shown in SEQ ID NO.8 of rubella virus is used to increase, a group or more the combination of primer of genome nucleic acid fragment shown in SEQ ID NO.9 of Coxsackie B virus 1 type is used for increasing.

Preferably, the primer of test kit of the present invention is selected from following 9 groups of primers:

Preferably, test kit of the present invention also comprises the capture probe shown in nucleotide sequence SEQ ID NO.37.

More preferably, test kit of the present invention also comprises the following at least a detection probes corresponding with primer:

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 influenza virus A hypotype, influenza virus B hypotype, human parainfluenza virus's 1 hypotype, human parainfluenza virus's 2 hypotypes, human parainfluenza virus's 3 hypotypes, human parainfluenza virus's 4 hypotypes, respiratory syncytial virus, rubella virus, Coxsackie virus is chosen in search according to NCBI, and they are respectively:

Influenza virus A hypotype stromatin 1 gene order, its GENEBANK accession number is EU716171, choose sequence high conservative part as target nucleic acid sequence, the target sequence that the present invention chooses is stromatin 1 gene 32-274, and its nucleotide sequence is shown in SEQ ID NO.1;

The 8th sections gene order of influenza virus B hypotype, the GENEBANK accession number is CY033960, and the target nucleic acid sequence that the present invention chooses is the 716-839 of the 8th sections gene, and its nucleotide sequence is shown in SEQID NO.2;

The nucleocapsid protein gene order of human parainfluenza virus's 1 hypotype, GENEBANK accession number are U70948, and the target nucleic acid sequence that the present invention chooses is the 858-1051 of nucleocapsid protein gene, and its nucleotide sequence is shown in SEQ ID NO.3;

The nucleocapsid protein gene order of human parainfluenza virus's 2 hypotypes, GENEBANK accession number are AF213352, and the target nucleic acid sequence that the present invention chooses is the 515-689 of nucleocapsid protein gene, and its nucleotide sequence is shown in SEQ ID NO.4;

The nucleocapsid protein gene order of human parainfluenza virus's 3 hypotypes, GENEBANK accession number are M18760, and the target nucleic acid sequence that the present invention chooses is the 1076-1200 of nucleocapsid protein gene, and its nucleotide sequence is shown in SEQ ID NO.5;

Human parainfluenza virus's 4 hypotype phosphoric acid protein gene sequences, the GENEBANK accession number is M55976, and the target nucleic acid sequence that the present invention chooses is the 951-1088 of phosphoric acid protein gene, and its nucleotide sequence is shown in SEQID NO.6.

Respiratory syncytial virus N gene order, GENEBANK accession number are D00736, and the target nucleic acid sequence that the present invention chooses is the 1681-1809 of N gene, and its nucleotide sequence is shown in SEQ ID NO.7.

Glycoprotein E 1 gene order of rubella virus, the GENEBANK accession number is M15240, and the target nucleic acid sequence that the present invention chooses is the 8788-8891 of glycoprotein E 1 gene, and its nucleotide sequence is shown in SEQ IDNO.8.

The genome sequence of Coxsackie B virus 1 type, GENEBANK accession number are M16560, and the target nucleic acid sequence that the present invention chooses is the genomic 457-600 of Coxsackie B virus 1 type, and its nucleotide sequence is shown in SEQ ID NO.9.

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.37; The detection probes of primer and correspondence is as follows:

The 1st group: the upstream primer nucleotide sequence of the nucleic acid fragment of influenza virus A hypotype stromatin 1 shown in SEQ ID NO.1 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 2nd group: the upstream primer nucleotide sequence of the nucleic acid fragment of the 8th sections gene shown in SEQ ID NO.2 of the influenza virus B hypotype 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 3rd group: the upstream primer nucleotide sequence of the nucleic acid fragment of human parainfluenza virus's 1 hypotype nucleocapsid protein gene shown in SEQ ID NO.3 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 4th group: the upstream primer nucleotide sequence of the nucleic acid fragment of human parainfluenza virus's 2 hypotype nucleocapsid protein genes shown in SEQ ID NO.4 that be used to increase shown in SEQ ID NO.19, the downstream primer nucleotide sequence shown in SEQ ID NO.20, detection probes is shown in SEQ ID NO.21;

The 5th group: the upstream primer nucleotide sequence of the nucleic acid fragment of nucleocapsid protein gene shown in SEQ ID NO.5 of human parainfluenza virus's 3 hypotypes that are used to increase shown in SEQ ID NO.22, the downstream primer nucleotide sequence shown in SEQ ID NO.23, detection probes is shown in SEQ ID NO.24;

The 6th group: the upstream primer nucleotide sequence of human parainfluenza virus's 4 hypotype phosphoric acid protein genes nucleic acid fragment shown in SEQ ID NO.6 that is used to increase shown in SEQ ID NO.25, the downstream primer nucleotide sequence shown in SEQ ID NO.26, detection probes is shown in SEQ ID NO.27.

The 7th group: the upstream primer nucleotide sequence of the respiratory syncytial virus N gene nucleic acid fragment shown in SEQ ID NO.7 that is used to increase shown in SEQ ID NO.28, the downstream primer nucleotide sequence shown in SEQID NO.29, detection probes is shown in SEQ ID NO.30.

The 8th group: the upstream primer nucleotide sequence of glycoprotein E 1 gene nucleic acid fragment shown in SEQ ID NO.8 of the rubella virus that is used to increase shown in SEQ ID NO.31, the downstream primer nucleotide sequence shown in SEQ ID NO.32, detection probes is shown in SEQ ID NO.33.

The 9th group: the upstream primer nucleotide sequence of genome nucleic acid fragment shown in SEQ ID NO.9 of Coxsackie B virus 1 type that is used to increase shown in SEQ ID NO.34, the downstream primer nucleotide sequence shown in SEQ ID NO.35, detection probes is shown in SEQ ID NO.36.

Embodiment 2

Prepare test kit of the present invention.Consist of:

1, amplification system

Every group of 10uM of primer (9 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 the nucleocapsid protein gene, human parainfluenza virus's 4 hypotype phosphoric acid protein genes, respiratory syncytial virus N gene, glycoprotein E 1 gene of rubella virus, the genome of Coxsackie B virus 1 type of nucleocapsid protein gene, human parainfluenza virus's 3 hypotypes of nucleocapsid protein gene, human parainfluenza virus's 2 hypotypes of the 8th sections gene, human parainfluenza virus's 1 hypotype of influenza virus A hypotype stromatin 1, the influenza virus B hypotype of 5pM synthetic.

2, detection architecture

Detection probes (9 groups) 26 μ M by digoxigenin labeled

Capture probe (biotin labeling) 26 μ M

Cleaning buffer solution 1 x TBS

Hybridization buffer 10mg/ml BSA, 50mM Tris-HCl (pH 7.5)

Detect damping fluid 1 x 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;

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 adds the 1ml guanidinium isothiocyanate, adds 1ml TRIZOL behind the mixing again, and the vibration mixing was placed 5 minutes in the ice bath.Add the 350ul chloroform, the mixing that 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) mixing.Place 1h for-20 ℃, 4 ℃, 12000r/ minute centrifugal 20 minutes, precipitation was total RNA.Add 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 obtained the determined nucleic acid sample, and-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=1:500 dilution), 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:

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 is centrifugal, and 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.Add the 350ul chloroform, the mixing that fully vibrates, after the static layering, immediately in 4 ℃, 12000r/ minute is centrifugal 20 minutes.Supernatant is transferred to another centrifuge tube, adds isopyknic Virahol (4 ℃ of precoolings) mixing.Place 1h for-20 ℃, 4 ℃, centrifugal 20 minutes of 12000r/min, precipitation is total RNA.Add the 0.5ml75% washing with alcohol, 4 ℃, centrifugal 10 minutes of 12000r/min carefully outwells ethanol, and room temperature was placed 10 minutes, added an amount of DEPC treated water dissolution precipitation, promptly obtained the determined nucleic acid sample, and-80 ℃ of preservations are standby.

2, NASBA amplification

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=1:500 configuration) that 100 μ l detect damping fluid and detect damping fluid, at room temperature incubation is 30 minutes, with 250 μ l 1x TBS, pH7.4 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 influenza virus A hypotype

1, nucleic acid amplification

Get 1ml influenza virus A hypotype standard substance and add the 1ml guanidinium isothiocyanate, add 1mlTRIZOL behind the mixing again, the vibration mixing was placed 5 minutes in the ice bath.Add the 350ul chloroform, the mixing that 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) mixing.Place 1h for-20 ℃, 4 ℃, centrifugal 20 minutes of 12000r/min, precipitation is total RNA.Add 0.5ml 75% washing with alcohol, 4 ℃, centrifugal 10 minutes of 12000r/min carefully outwells ethanol, and room temperature was placed 10 minutes, added an amount of DEPC treated water dissolution precipitation, promptly obtained the determined nucleic acid sample, measured 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, influenza virus A hypotype 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.

Influenza virus A hypotype RNA

Primer (the 1st 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

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.1, and is consistent with influenza virus A hypotype stromatin 1 gene order 32-274 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 (the 1st group) 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=1:500 configuration), 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 A subtype influenza virus (AIV)

Sample	AIV	AIV	AIV	AIV	AIV	AIV	AIV	AIV	No RNA enzyme water	No RNA enzyme water
Sample	AIV	AIV	AIV	AIV	AIV	AIV	AIV	AIV	No RNA enzyme water	No RNA enzyme water	Concentration (pM)	10 ^-9	10 ^-9	10 ^-10	10 ^-10	10 ^-11	10 ^-11	10 ^-12	10 ^-12
OD value 405nm	3.087	3.107	1.304	1.432	0.839	0.870	0.361	0.211	0.210	0.223	Concentration (pM)	10 ^-9	10 ^-9	10 ^-10	10 ^-10	10 ^-11	10 ^-11	10 ^-12	10 ^-12

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 influenza virus A hypotype is 10 ^-11PM.

Embodiment 6: the sensitivity experiment that detects other pathogenic agent

With reference to embodiment 5, influenza virus B hypotype, human parainfluenza virus's 1 hypotype, human parainfluenza virus's 2 hypotypes, human parainfluenza virus's 3 hypotypes, human parainfluenza virus's 4 hypotypes, respiratory syncytial virus, rubella virus and the Coxsackie virus of preparation different concns, carry out NASBA amplification back with its corresponding primer and measure the amplified production sequence, its Nucleotide is consistent with target gene.Detect with corresponding capture probe and detection probes, detected every kind of pathogenic agent minimum concentration all can reach 10 ^-11PM.

Embodiment 7: detect the primer specificity test of influenza virus A hypotype

The 38-41 position of the gene fragment of influenza virus A hypotype stromatin 1 gene shown in SEQ ID NO.1 is suddenlyd change, nucleotide sequence after the sudden change is shown in SEQ ID NO.38, in order to narrate conveniently, be referred to as influenza virus A hypotype stromatin 1 gene fragment interference sequence.

Get isocyatic embodiment 5 influenza virus A hypotype standard substance, above-mentioned interference sequence and dna sequence dna, get one of them or mixing in twos or three kinds of mixing and be mixed with 7 kinds of different testing samples altogether, every kind of testing sample is provided with multiple pipe, 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 the negative control of two no RNA enzyme water is set simultaneously, 41 ℃ of water-baths 95 minutes.Nucleic acid amplification product is used for subsequent detection.

Testing sample 800 μ M

Primer (the 1st 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

Carry out SDS-PAGE electrophoretic analysis, the result shows that the reaction tubes that has only mixed influenza virus A hypotype standard substance has amplified production, 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.1 ' connected and capture probe complementary sequence.

Embodiment 8: the specificity experiment of other primers

With reference to embodiment 7, the nucleic acid fragment of the 8th sections gene shown in SEQ ID NO.2 with the influenza virus B hypotype, the nucleic acid fragment of the nucleocapsid protein gene of human parainfluenza virus's 1 hypotype shown in SEQ ID NO.3, the nucleic acid fragment of the nucleocapsid protein gene of human parainfluenza virus's 2 hypotypes shown in SEQ ID NO.4, the nucleic acid fragment of the nucleocapsid protein gene of human parainfluenza virus's 3 hypotypes shown in SEQ ID NO.5, the nucleic acid fragment of human parainfluenza virus's 4 hypotype phosphoric acid protein genes shown in SEQ ID NO.6, the nucleic acid fragment of respiratory syncytial virus N gene shown in SEQ ID NO.7, the nucleic acid fragment of glycoprotein E 1 gene shown in SEQ ID NO.8 of rubella virus, the interference sequence and the target nucleic acid fragment that form behind the several base random mutations of the nucleic acid fragment of genome shown in SEQID NO.9 of Coxsackie B virus 1 type, people DNA carries out the specificity experiment, promptly get one of them or mixing in twos or three kinds of mixing and be mixed with 7 kinds of different testing samples altogether, increase with corresponding primer at different reaction tubess respectively, SDS-PAGE electrophoretic analysis shows only has the reaction tubes that has mixed target nucleic acid that amplified production is arranged, 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>MP090691

<160>38

<170>PatentIn version 3.3

<210>1

<211>242

<212>DNA

＜213〉influenza virus A hypotype stromatin 1 gene 32-274

<400>1

<210>2

<211>124

<212>DNA

＜213〉the 8th sections gene 716-839 of influenza virus B type

<400>2

<210>3

<211>194

<212>DNA

＜213〉858-1051 of human parainfluenza virus's 1 hypotype nucleocapsid protein gene

<400>3

<210>4

<211>175

<212>DNA

＜213〉515-689 of human parainfluenza virus's 2 hypotype nucleocapsid protein genes

<400>4

<210>5

<211>125

<212>DNA

＜213〉1076-1200 of human parainfluenza virus's 3 hypotype nucleocapsid protein genes

<400>5

<210>6

<211>138

<212>DNA

＜213〉951-1088 of human parainfluenza virus's 4 hypotype phosphoric acid protein genes

<400>6

<210>7

<211>129

<212>DNA

＜213〉1681-1809 of respiratory syncytial virus N gene

<400>7

<210>8

<211>104

<212>DNA

＜213〉8788-8891 of rubella virus glycoprotein E 1 gene

<400>8

<210>9

<211>144

<212>DNA

＜213〉the genomic 457-600 of Coxsackie B virus 1 type

<400>9

<210>10

<211>43

<212>DNA

＜213〉primer I nfA-F

<400>10

<210>11

<211>55

<212>DNA

＜213〉primer I nfA-R

<400>11

<210>12

<211>23

<212>DNA

＜213〉probe I nfA-P

<400>12

<210>13

<211>45

<212>DNA

＜213〉primer I nfB-F

<400>13

<210>14

<211>56

<212>DNA

＜213〉primer I nfB-R

<400>14

<210>15

<211>23

<212>DNA

＜213〉probe I nfB-P

<400>15

<210>16

<211>44

<212>DNA

＜213〉primer HPIV1-F

<400>16

<210>17

<211>56

<212>DNA

＜213〉primer HPIV1-R

<400>17

<210>18

<211>25

<212>DNA

＜213〉probe HPIV1-P

<400>18

<210>19

<211>43

<212>DNA

＜213〉primer HPIV2-F

<400>19

<210>20

<211>55

<212>DNA

＜213〉primer HPIV2-R

<400>20

<210>21

<211>25

<212>DNA

＜213〉probe HPIV2-P

<400>21

<210>22

<211>44

<212>DNA

＜213〉primer HPIV3-F

<400>22

<210>23

<211>57

<212>DNA

＜213〉primer HPIV3-R

<400>23

<210>24

<211>25

<212>DNA

＜213〉probe HPIV3-P

<400>24

<210>25

<211>46

<212>DNA

＜213〉primer HPIV4-F

<400>25

<210>26

<211>56

<212>DNA

＜213〉primer HPIV4-R

<400>26

<210>27

<211>25

<212>DNA

＜213〉probe HPIV4-P

<400>27

<210>28

<211>46

<212>DNA

＜213〉primer RSV-F

<400>28

<210>29

<211>55

<212>DNA

＜213〉primer RSV-R

<400>29

<210>30

<211>23

<212>DNA

＜213〉probe RSV-P

<400>30

<210>31

<211>42

<212>DNA

＜213〉primer RV-F

<400>31

<210>32

<211>56

<212>DNA

＜213〉primer RV-R

<400>32

<210>33

<211>24

<212>DNA

＜213〉probe RV-P

<400>33

<210>34

<211>42

<212>DNA

＜213〉primer Cox-F

<400>34

<210>35

<211>51

<212>DNA

＜213〉primer Cox-R

<400>35

<210>36

<211>23

<212>DNA

＜213〉probe Cox-P

<400>36

<210>37

<211>20

<212>DNA

＜213〉capture probe

<400>37

<210>38

<211>242

<212>DNA

＜213〉influenza virus A hypotype stromatin 1 gene fragment interference sequence

<400>38

Claims

1, a kind of method that the infectious disease pathogens that may be present in the biological sample are detected comprises:

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 9 groups of oligonucleotide sequences below described step (i) is used is increased as primer:

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.37, 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:

7, a kind of test kit that the infectious disease pathogens that may be present in the biological sample are detected, it comprises the primer of shown in the SEQ ID NO.1 nucleic acid fragment of being used to increase, the primer of shown in SEQ IDNO.2 nucleic acid fragment is used to increase, the primer of shown in SEQ ID NO.3 nucleic acid fragment is used to increase, the primer of shown in SEQ ID NO.4 nucleic acid fragment is used to increase, the primer of shown in SEQ ID NO.5 nucleic acid fragment is used to increase, the primer of shown in SEQ ID NO.6 nucleic acid fragment is used to increase, the primer of shown in SEQ ID NO.7 nucleic acid fragment is used to increase, a group or more the combination of primer of the primer of shown in SEQ ID NO.8 nucleic acid fragment and shown in the SEQ ID NO.9 nucleic acid fragment of being used to increase is used for increasing.

8, test kit as claimed in claim 7, described primer is selected from:

9, test kit according to claim 8 is characterized in that, also comprises the capture probe shown in nucleotide sequence SEQ ID NO.37.

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: