CN101545009B - Method for detecting infectious disease pathogens and kit - Google Patents
Method for detecting infectious disease pathogens and kit Download PDFInfo
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- CN101545009B CN101545009B CN200910083896.3A CN200910083896A CN101545009B CN 101545009 B CN101545009 B CN 101545009B CN 200910083896 A CN200910083896 A CN 200910083896A CN 101545009 B CN101545009 B CN 101545009B
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Abstract
The invention discloses a method for detecting infectious disease pathogens possibly existing in a biological sample. The infectious disease pathogens comprise chlamydia psittaci, pneumocystis carinii, leptospira, Q fever rickettsiosis and brucellosis. 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 simultaneously detect various infectious disease pathogens, and is suitable for early diagnosis of respiratory infectious diseases.
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, easily the pathogenic agent of transmissible disease is detected, be the key of controlling disease transmission.Although set up the Monitoring systems of transmissible disease on internal and international, existing detection means respectively has limitation.The corresponding antibody of Serological testing utilization is combined and pathogenic agent detected with antigen, this method diagnosis fast, simply, easy handling, but must prepare for detecting after the antibody of this pathogenic agent, especially in the situation that also there is no antibody sources, not be suitable for the early diagnosis of transmissible disease.
Pathogen isolation method is by directly pathogenic agent being carried out to separation and Culture, detects and identifies pathogenic agent.This method is highly sensitive, high specificity, but complicated operation, time-consuming (at least will spend week age), very high to laboratory Biosafety conditional request, thus be difficult in epidemic situation generation field by using, and also not every pathogenic agent can obtain by 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 whole reaction process, but exist DNA to pollute the high problem of false positive rate causing always, and because its threshold value is not obvious, therefore detected result gray area is wider, cannot stdn, and because instrument requires high, have high input, operator are required high, need to carry out professional skill training, also limited the application of Multiple detection.The method still cannot 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, the nucleic acid amplification technologies based on enzyme reaction.The reaction system of this technology comprises ThermoScript II, ribonuclease H, phage t7 ribonucleic acid polymerase and two specially designed Oligonucleolide primers, 3 ' end of its upstream primer 3 ' end and template is complementary, the promoter sequence of the RNA polymerase that depends on DNA that 5 ' end contains phage t7, downstream primer 3 ' end sequence is consistent with 5 ' end sequence of template, and 5 ' end contains the sequence with capture probe complementation.At amplification initial period, after upstream primer be combined with just RNA template, under the effect of ThermoScript II, synthesize and the Antisense cDNA of target RNA complementation, originally RNA template by RnaseH, degraded.Then downstream primer and cDNA hybridization, synthetic double chain cDNA under the effect of the archaeal dna polymerase characteristic of ThermoScript II, 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 sense-rna chain target sequence complementation.So repeatedly, RNA copy number is constantly amplified in circulation.Simultaneously, because another end of double-stranded cDNA has also been integrated the sequence with capture probe complementation, the combination capture probe that the complementary RNA therefore producing again can be special, for next step detection.
It is that NASBA amplified production is combined with capture probe one end first specifically that enzyme connects oligonucleotide capture technique principle, the latter's the other end can be fixedly connected on microwell plate, then add pathogen specific detection probes and reaction substrate, detect the ratio chrominance signal producing, this detection reading is directly proportional to the amplified production amount of RNA, with this, detect NASBA amplified production total amount, the infection conditions of judgement viral template.This method is simple to operate, and high specificity is applicable to the rapid detection of a large amount of samples.
The symptoms such as the high heat of transmissible disease that chlamydia psittaci, Pneumocystis carinii, Leptospira, the hot rickettsia of Q and brucella cause, lassitude, sore all over are obvious, main through directly contact or droplet transmission, 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 above-mentioned five kinds of infectious disease pathogens simultaneously, also there is no corresponding clinical detection reagent box listing.
Summary of the invention
The present invention seeks to overcome the defect that prior art can not detect multiple infectious disease pathogenic agent simultaneously, a kind of method that the infectious disease pathogens that may be present in biological sample are detected is provided, described infectious disease pathogens comprise chlamydia psittaci, Pneumocystis carinii, Leptospira, the hot rickettsia of Q and brucella, 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 as shown in SEQ ID NO.1 of chlamydia psittaci endosome membranin A gene;
The nucleic acid fragment as shown in SEQ ID NO.2 of the main surface glycoprotein A gene of Pneumocystis carinii;
The nucleic acid fragment as shown in SEQ ID NO.3 of Leptospira gyrase B subunit gene;
The nucleic acid fragment as shown in SEQ ID NO.4 of the hot rickettsia outer membrane protein gene of Q;
The nucleic acid fragment as shown in SEQ ID NO.5 of brucella outer membrane protein omp2b gene;
(ii) with probe, detect a kind of specific hybrid in described probe and step (i) amplified production.
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.
In step of the present invention (ii), preferably the hybridization of described probe and step (i) gained amplified production is carried out on solid support.
Can stationary probe and one or more of nucleic acid fragment, for example, be fixed on Sptting plate.Term " solid support " refers to and keeps the solid substrate that oligonucleotide probe under its hybridization characteristic prerequisite can coupling, and conventionally, solid substrate is nylon end, cellulose membrane, microballon, chip or Sptting plate.Before fixing, can modify probe, to promote fixing or raising hybridization efficiency.Such modification comprises homopolymeric tailing, with NH
2group or vitamin H, hapten conjugation.
Or probe and nucleic acid fragment are all unfixed, 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 is used for the nucleic acid fragment increasing as shown in SEQ ID NO.1, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.6, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.7;
The 2nd group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.2, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.9, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.10;
The 3rd group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.3, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.12, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.13;
The 4th group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.4, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.15, and downstream primer nucleotide sequence is as shown in SEQ ID NO.16;
The 5th group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.5, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.18, and downstream primer nucleotide sequence is as shown in SEQ ID NO.19.
Above-mentioned the 1st group to the 5th group primer be corresponding chlamydia psittaci, Pneumocystis carinii, Leptospira, the hot rickettsia of Q, brucella respectively.
In actual applications, also having a kind of may be any one or more than one the combination of using in above-mentioned primer group, increase and detect a certain or several in chlamydia psittaci, Pneumocystis carinii, Leptospira, the hot rickettsia of Q and brucella, as using the sequence of the 2nd group and the 4th group to carry out Pneumocystis carinii and the rickettsial detection of Q heat etc.
The capture probe that the present invention also provides described step (ii) to use, its nucleotide sequence is as shown in SEQ IDNO.21, and what it was direct or indirect is connected on solid support, and hybridizes with step (i) gained amplified production.
The present invention also provides the detection probes of using in step (ii):
Sequence 1, as shown in SEQ ID NO.8, detects the nucleic acid fragment as shown in SEQ ID NO.1;
Sequence 2, as shown in SEQ ID NO.11, detects the nucleic acid fragment as shown in SEQ ID NO.2;
Sequence 3, as shown in SEQ ID NO.14, detects the nucleic acid fragment as shown in SEQ ID NO.3;
Sequence 4, as shown in SEQ ID NO.17, detects the nucleic acid fragment as shown in SEQ ID NO.4;
Sequence 5, as shown in SEQ ID NO.20, detects the nucleic acid fragment as shown in SEQ ID NO.5.
Above-mentioned sequence 1～5 is the corresponding hot rickettsia of chlamydia psittaci, Pneumocystis carinii, Leptospira, Q, the brucella detecting after amplification respectively.
Detection probes can detect with multiple biological method.Preferably, described detection probes, by vitamin H or digoxigenin labeled, adds substrate, reads absorbance carry out result judgement through termination reaction step by spectrophotometer.
The present invention preferably embodiment is to carry out at one time the described detecting step of step (ii) by a plurality of reaction tubess, and the detection probes that each reaction tubes is used should detect the existence of a certain pathogenic agent to a kind of in sequence 5 and with the primer pair that step (i) is used jointly for sequence 1.
The absorbancy result treatment of aforesaid method can be taked following scheme: a tested K negative control sample, get its absorbance, determine as follows threshold value: m+K * SD, the negative contrast absorbancy of m arithmetical av, the negative contrast absorbancy of SD standard deviation, K determines according to different experimental conditions, as the colony's number or the sample number that detect.Detection reading is greater than threshold value and is judged to be detected result " positive "; Detection reading is less than threshold value and is judged to be detected result " feminine gender ".
Term of the present invention " primer " refers to the single stranded oligonucleotide sequence as the initiation site of synthetic primer extension products, and it is complementary with nucleic acid chains to be copied, and length and sequence must be suitable for the synthetic of extension products.
Term of the present invention " probe " refers to single stranded oligonucleotide, for nucleic acid fragment specific hybrid." specific hybrid " refers to that the whole region of described probe and nucleic acid or a part are forming duplex under specific experiment condition, and under these conditions, described probe not with detected sample in other nucleic acid of existing or other regions of nucleic acid form duplexs.
After probe and nucleic acid fragment hybridization, can be by the mode of any appropriate, the detection of hybridizing, the label probe that for example can detect and/or nucleic acid fragment, for auxiliary detection, preferably 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 biological sample are detected, it comprises the primer for the chlamydia psittaci endosome membranin A gene nucleic acid fragment as shown in SEQ ID NO.1 that increases, the primer of main surface glycoprotein A gene nucleic acid fragment as shown in SEQ IDNO.2 for the Pneumocystis carinii of increasing, primer for the Leptospira gyrase B subunit gene nucleic acid fragment as shown in SEQID NO.3 that increases, for the primer of the hot rickettsia outer membrane protein gene of the Q nucleic acid fragment as shown in SEQID NO.4 that increases with for a group or more combination of the primer of the brucella outer membrane protein omp2b gene nucleic acid fragment as shown in SEQ ID NO.5 that increases.
Preferably, the primer of test kit of the present invention is selected from following 5 groups of primers:
The 1st group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.1, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.6, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.7;
The 2nd group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.2, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.9, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.10;
The 3rd group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.3, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.12, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.13;
The 4th group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.4, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.15, and downstream primer nucleotide sequence is as shown in SEQ ID NO.16;
The 5th group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.5, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.18, and downstream primer nucleotide sequence is as shown in SEQ ID NO.19.
Preferably, test kit of the present invention also comprises the capture probe as shown in nucleotide sequence SEQ ID NO.21.
More preferably, test kit of the present invention also comprises the detection probes that following at least one and primer pair are answered:
Sequence 1, as shown in SEQ ID NO.8, detects the nucleic acid fragment as shown in SEQ ID NO.1;
Sequence 2, as shown in SEQ ID NO.11, detects the nucleic acid fragment as shown in SEQ ID NO.2;
Sequence 3, as shown in SEQ ID NO.14, detects the nucleic acid fragment as shown in SEQ ID NO.3;
Sequence 4, as shown in SEQ ID NO.17, detects the nucleic acid fragment as shown in SEQ ID NO.4;
Sequence 5, as shown in SEQ ID NO.20, detects the nucleic acid fragment as shown in SEQ ID NO.5.
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 forming; For probe is attached to the component on solid support.
The method that the infectious disease pathogens that may be present in biological sample are detected of the present invention, the advantage of contrast prior art is:
(1) detection speed is fast, and efficiency is high, can be as required, and double, triple or Multiple Combination detects multiple infective pathogen body simultaneously at random, also can detect single pathogenic agent;
(2) highly sensitive, it is 10 that NASBA can detect viral Cmin
-11pM, higher than the susceptibility of classical pathogen separation method;
(3) high specificity, because external double-stranded DNA is without T7 promoter sequence, can not be amplified, and this has just significantly improved the specificity of NASBA reaction; Due to the primer in amplification procedure and the probe dual function in testing process, again guaranteed the specificity of pathogen detection, and the reaction conditions of NASBA is gentle, and shorter than the time of PCR reaction needed, therefore transcribe more faithful to template, further reduced mispairing rate;
(4) simple to operate, 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 early stage rapid detection of great communicate illness that FUO causes, the random combine of multiple pathogens detects and the check of a large amount of samples.
(6) applied widely, sampling is simple.In sample, the pollution of DNA, heparin, EDTA, Citrate trianion, oxyphorase, albumin and lipid etc. will can not impact result, thereby is applicable to various samples to be checked as brush,throat, ight soil, blood etc.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment are only not used in and limit the scope of the invention for the present invention is described, 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 in embodiment below, conventionally according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
Design and prepare primer, probe sequence.
According to NCBI search, choose chlamydia psittaci, Pneumocystis carinii, Leptospira, the hot rickettsia of Q, brucellar specific gene sequence, they respectively:
Chlamydia psittaci endosome membranin A gene order, GENEBANK accession number is DQ117471, choose sequence high conservative part as target nucleic acid sequence, the target sequence that the present invention chooses is membranin A gene 317-472, and its nucleotide sequence is as shown in SEQ ID NO.1;
The main surface glycoprotein A gene sequence of Pneumocystis carinii, GENEBANK accession number is AF033209, and the target nucleic acid sequence that the present invention chooses is main surface glycoprotein A gene 2895-3010, and its nucleotide sequence is as shown in SEQ ID NO.2;
Leptospira gyrase B subunit gene sequence, GENEBANK accession number is AY896758, and the target nucleic acid sequence that the present invention chooses is gyrase B subunit gene 181-331, and its nucleotide sequence is as shown in SEQ ID NO.3;
The hot rickettsia outer membrane protein gene of Q sequence, GENEBANK accession number is AF318146, and the target nucleic acid sequence that the present invention chooses is outer membrane protein gene 703-883, and its nucleotide sequence is as shown in SEQ IDNO.4;
Brucella outer membrane protein omp2b gene order, GENEBANK accession number is EF534310, and the target nucleic acid sequence that the present invention chooses is outer membrane protein omp2b gene 767-1002, and its nucleotide sequence is as shown in SEQ ID NO.5;
Analyze the information of above-mentioned virus-specific gene, eliminating through carry out between primer/interdimers of 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 for above-mentioned pathogen detection.
The nucleotide sequence of capture probe is as shown in SEQ ID NO.21; The detection probes of primer and correspondence is as follows:
The 1st group: for the upstream primer nucleotide sequence of the nucleic acid fragment of chlamydia psittaci endosome membranin A gene as shown in SEQ ID NO.1 that increase as shown in SEQ ID NO.6, downstream primer nucleotide sequence as shown in SEQ ID NO.7, detection probes is as shown in SEQ ID NO.8;
The 2nd group: the upstream primer nucleotide sequence of the nucleic acid fragment for the main surface glycoprotein A gene of the fowl Pneumocystis carinii of increasing as shown in SEQ ID NO.2 as shown in SEQ ID NO.9, downstream primer nucleotide sequence as shown in SEQ ID NO.10, detection probes is as shown in SEQ ID NO.11;
The 3rd group: for the upstream primer nucleotide sequence of the nucleic acid fragment of Leptospira gyrase B subunit gene as shown in SEQ ID NO.3 that increase as shown in SEQ ID NO.12, downstream primer nucleotide sequence as shown in SEQ ID NO.13, detection probes is as shown in SEQ ID NO.14;
The 4th group: the upstream primer nucleotide sequence of the nucleic acid fragment for the hot rickettsia outer membrane protein gene of the Q that increases as shown in SEQ ID NO.4 as shown in SEQ ID NO.15, downstream primer nucleotide sequence as shown in SEQ ID NO.16, detection probes is as shown in SEQ ID NO.17;
The 5th group: for the upstream primer nucleotide sequence of the nucleic acid fragment of brucella outer membrane protein omp2b gene as shown in SEQ ID NO.5 that increase as shown in SEQ ID NO.18, downstream primer nucleotide sequence as shown in SEQ ID NO.19, detection probes is as shown in SEQ ID NO.20;
Embodiment 2
Prepare test kit of the present invention.Consist of:
1, amplification system
Every group of 10uM of primer (5 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 without RNA enzyme water
Positive control is the chlamydia psittaci endosome membranin A gene order of 5pM synthetic, the main surface glycoprotein A gene sequence of Pneumocystis carinii, Leptospira gyrase B subunit gene sequence, the hot rickettsia outer membrane protein gene of Q sequence, brucella outer membrane protein omp2b gene order.
2, detection system
Detection probes (5 groups, by digoxigenin labeled) 26 μ M
Capture probe (biotin labeling) 26 μ M
Cleaning buffer solution 1xTBS
Hybridization buffer 10mg/ml BSA, 50mM Tris-HCl (pH 7.5)
Detect damping fluid 1xTBS: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 without RNA enzyme water;
Positive control is the chlamydia psittaci endosome membranin A gene order of 5pM synthetic, the main surface glycoprotein A gene sequence of Pneumocystis carinii, Leptospira gyrase B subunit gene sequence, the hot rickettsia outer membrane protein gene of Q sequence, brucella outer membrane protein omp2b gene order.
Embodiment 3:
The method that the infectious disease pathogens that may be present in biological sample are detected is also the using method of test kit of the present invention simultaneously.
1, nucleic acid extraction
Get nasopharyngeal secretions sample to be measured, centrifuging and taking supernatant, add 1ml guanidinium isothiocyanate, after mixing, add again 1ml TRIZOL, vibration mixes, in ice bath, place 5 minutes. add 350ul chloroform, fully vibration mixes, after static layering, immediately in 4 ℃, centrifugal 20 minutes of 12000r/min. supernatant is transferred to another centrifuge tube, add isopyknic Virahol (4 ℃ of precoolings) and mix .-20 ℃ of placement 1h, 4 ℃, 12000r/ minute centrifugal 20 minutes, precipitation is that total RNA. adds 0.5ml 75% washing with alcohol, 4 ℃, 12000r/ minute centrifugal 10 minutes, carefully outwell ethanol, room temperature is placed 10 minutes, add appropriate DEPC treated water dissolution precipitation, obtain determined nucleic acid sample,-80 ℃ save backup.
2, NASBA amplified reaction
20 μ l NASBA amplification reaction systems shown in being formulated as follows in sterilizing centrifuge tube at 0.5ml without RNA enzyme, 41 ℃ of incubations 90 minutes, nucleic acid amplification product was 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, 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, after mixing with 43 μ l hybridization buffers, add be coated with streptavidin microwell plate (purchased from Thermo fisher scientific) each independently in hole, 45 ℃ of incubations 1 hour, with 250 μ l 1x TBS, pH 7.4 cleans aperture three times.Incline after solution at every turn and will pat dry microwell plate, every hole adds the mixture (by detecting concentrated solution: detect damping fluid=1: 500 dilute) that 100 μ l detect damping fluid and detect concentrated solution, detect concentrated solution purchased from Sigma, P/N N7653, at room temperature incubation is 30 minutes, with 250 μ l 1xTBS, pH 7.4 cleans aperture three times, incline after solution at every turn and will pat dry microwell plate, micropore adds 100 μ l substrates, lucifuge room temperature incubation 5 minutes, 100 μ l stop buffers also shake color development stopping reaction gently, microwell plate and microwell plate sheet frame are put into standard 96 hole microwell plate spectrophotometers and read 405nm absorbancy.100 μ l substrates add 100 μ l stop buffer references as a setting.
4, Analysis of test results
Above-mentioned absorbancy result treatment is taked following scheme: test 8 its absorbances of negative control sample determination, determine as follows threshold value: m+8 * SD, the negative contrast absorbancy of m arithmetical av, the negative contrast absorbancy of SD standard deviation.Threshold value is 0.15+0.035 * 8=0.43.Detection reading is greater than threshold value and is judged to be " positive "; Detection reading is less than threshold value and is judged to be " feminine gender ".
Embodiment 4:
The method that the infectious disease pathogens that may be present in biological sample are detected is also 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, mix latter 4 ℃, 3000r/ minute centrifugal, 10 minutes, get supernatant, add 1ml guanidinium isothiocyanate, after mixing, add again 1ml TRIZOL, vibration mixes, in ice bath, place 5 minutes. add 350ul chloroform, fully vibration mixes, after static layering, immediately in 4 ℃, 12000r/ minute is centrifugal 20 minutes. supernatant is transferred to another centrifuge tube, add isopyknic Virahol (4 ℃ of precoolings) and mix .-20 ℃ of placement 1h, 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 is placed 10 minutes, add appropriate DEPC treated water dissolution precipitation, obtain determined nucleic acid sample,-80 ℃ save backup.
2, NASBA amplification
20 μ l NASBA amplification reaction systems shown in being formulated as follows in sterilizing centrifuge tube at 0.5ml without RNA enzyme, 41 ℃ of incubations 90 minutes, nucleic acid amplification product was 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, 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, mix with 43 μ l hybridization buffers and add be coated with streptavidin microwell plate (purchased from Thermo fisher scientific company) each independently in hole, 45 ℃ of incubations 1 hour, with 250 μ l 1xTBS, pH 7.4 cleans aperture three times.Incline after solution at every turn and will pat dry microwell plate, every hole adds 100 μ l and detects damping fluid and the mixture (by detecting concentrated solution: detect damping fluid=1: 500 configure) that detects damping fluid, at room temperature incubation is 30 minutes, with 250 μ l 1xTBS, pH 7.4 cleans aperture three times, incline after solution at every turn and will pat dry microwell plate, micropore adds 100 μ l 10mM Tris/HCl, lucifuge room temperature incubation 5 minutes, add 100 μ l stop buffers and shake gently color development stopping reaction, microwell plate and microwell plate sheet frame are put into standard 96 hole microwell plate spectrophotometers and read 405nm absorbancy, use 100 μ l 10mM Tris/HCl to add 100 μ l stop buffer references as a setting.
While whether containing certain 3 kinds of pathogenic agent in detecting testing sample, only need when preparation amplification system, add this 3 kinds of primers that pathogenic agent is corresponding respectively at 3 reaction tubess, add this 3 kinds of probe solutions that pathogenic agent is corresponding during detection, other steps are same.
4, Analysis of test results
The absorbancy of measuring 10 known negative samples, threshold value is m (negative control absorbancy arithmetical av)+10 * SD (negative control absorbancy standard deviation), threshold value is generally 0.15+0.03 * 10=0.45.Detection reading is greater than threshold value and is judged to be detected result " positive "; Detection reading is less than threshold value and is judged to be detected result " feminine gender ".
Embodiment 5: detect brucellar sensitivity experiment
1, nucleic acid amplification
Get 1ml brucella standard substance and add 1ml guanidinium isothiocyanate, after mixing, add again 1mlTRIZOL, vibration mixes, in ice bath, place 5 minutes. add 350ul chloroform, fully vibration mixes, after static layering, immediately in 4 ℃, centrifugal 20 minutes of 12000r/min. supernatant is transferred to another centrifuge tube, add isopyknic Virahol (4 ℃ of precoolings) and mix .-20 ℃ of placement 1h, 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 is placed 10 minutes, add appropriate DEPC treated water dissolution precipitation, obtain determined nucleic acid sample, measure its concentration.
0.5ml without the sterilizing centrifuge tube of RNA enzyme in mark respectively, 20 μ lNASBA amplification reaction systems shown in being formulated as follows, and two negative controls are set simultaneously, brucella RNA is formulated as 10
-9pm, 10
-10pm, 10
-11pm, 10
-12the final concentration of pm, every kind of concentration arranges multiple pipe, usings without RNA enzyme water as negative control, 41 ℃ of water-baths 95 minutes.Nucleic acid amplification product is for subsequent detection.
The final concentration of NASBA amplification reaction system (20 μ l) is specially:
Brucella RNA
Primer (the 5th 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 as shown in SEQ ID NO.5, and 767-1002 is consistent with brucella outer membrane protein omp2b gene.
2, detection of nucleic acids
1) supporting to lay a microwell plate lath on sheet frame, this coated in microporous plate streptavidin (purchased from Thermo fisher scientific company).
2) in each micropore, add successively 43 μ l hybridization buffers, add 1 μ l detection probes solution (the 5th 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 sealed membrane and avoid evaporation, 45 ℃ of water-baths 60 minutes.
4) incline after solution, with 250 μ l 1xTBS, pH 7.4 cleans aperture five times, and cleans 30s at every turn, after the solution that inclines, pats dry microwell plate.
5) every hole adds the mixture (by detecting concentrated solution: damping fluid=1: 500 configure) of 100 μ l detection damping fluids and detection concentrated solution, firmly covers sealed membrane and avoids evaporation.
6) incubation 30 minutes at room temperature.
7) incline after solution, with 250 μ l 1xTBS, pH 7.4 cleans aperture five times, and cleans 30s at every turn, after the solution that at every turn inclines, pats dry microwell plate.
8) micropore adds 100 μ l connecting fluids (carefully avoiding introducing foam in hole).
9) lucifuge room temperature incubation is 5 minutes.
10) micropore adds 100 μ l stop buffers and shakes gently color development stopping reaction.
11) microwell plate and microwell plate sheet frame are put into standard 96 hole microwell plate spectrophotometers and read 405nm absorbancy.
3, result is as shown in table 1.
Table 1. the method for the invention detects the absorbance of different concns brucella (Bru)
| Sample | Bru | Bru | Bru | Bru | Bru | Bru | Bru | Bru | Without RNA enzyme water | Without RNA enzyme water |
| Concentration (pM) | 10 -9 | 10 -9 | 10 -10 | 10 -10 | 10 -11 | 10 -11 | 10 -12 | 10 -12 | ||
| OD value 405nm | 1.372 | 1.199 | 1.152 | 0.963 | 0.823 | 0.622 | 0.286 | 0.341 | 0.228 | 0.161 |
4, interpretation of result
Through spectrophotometer, read absorbance and carry out result 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), i.e. 0.17+0.04 * 7=0.45.Detection reading is greater than threshold value and is judged to be detected result " positive "; Detection reading is less than threshold value and is judged to be detected result " feminine gender ".Brucellar minimum concentration can be detected be 10 to the detection probes of the primer of the 5th group and the 5th group
-11pM.
Embodiment 6: the sensitivity experiment that detects other pathogenic agent
With reference to embodiment 5, the hot rickettsia of chlamydia psittaci, Pneumocystis carinii, Leptospira or Q of preparation different concns, carries out measuring amplified production sequence after NASBA amplification with its corresponding primer, and its nucleotide sequence is consistent with target gene.With corresponding capture probe and detection probes, detect, the minimum concentration detecting all can reach 10
-11pM.
Embodiment 7: detect brucellar primer specificity test
Gene fragment by brucella outer membrane protein omp2b gene as shown in SEQ ID NO.5 is suddenlyd change at random, nucleotide sequence after sudden change is as shown in SEQ ID NO.22, in order to narrate conveniently, be referred to as the interference sequence of brucella outer membrane protein omp2b gene.
Get brucella standard substance described in isocyatic embodiment 5, above-mentioned interference sequence and human DNA sequence, get one of them or mixing between two or three kinds of mixing and be mixed with altogether 7 kinds of different testing samples, every kind of testing sample arranges multiple pipe, 0.5ml without the sterilizing centrifuge tube of RNA enzyme in mark respectively, 20 μ l NASBA amplification reaction systems shown in being formulated as follows, and arrange two without the negative control of RNA enzyme water, 41 ℃ of water-baths 95 minutes simultaneously.Nucleic acid amplification product is for subsequent detection.
The final concentration of NASBA amplification reaction system (20 μ l) is specially:
Testing sample 800 μ M
Primer (the 5th 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
Carry out SDS-PAGE electrophoretic analysis, the reaction tubes that result has shown only to mix brucella standard substance has amplified production, get amplified production and entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to carry out sequential analysis, one section of sequence with capture probe complementation that its sequence has been 5 of nucleotide sequence as shown in SEQ ID NO.5 ' connect.
Embodiment 8: the specificity experiment of other primers
With reference to embodiment 7, nucleic acid fragment by chlamydia psittaci endosome membranin A gene as shown in SEQ ID NO.1, the nucleic acid fragment of the main surface glycoprotein A gene of Pneumocystis carinii as shown in SEQ ID NO.2, the nucleic acid fragment of Leptospira gyrase B subunit gene as shown in SEQ ID NO.3, the interference sequence and the target nucleic acid fragment that after several base random mutations of the nucleic acid fragment of the hot rickettsia outer membrane protein gene of Q as shown in SEQ ID NO.4, form, people DNA carries out specificity experiment, get one of them or mixing between two or three kinds of mixing and be mixed with altogether 7 kinds of different testing samples, at different reaction tubess, with corresponding primer, increase respectively, SDS-PAGE electrophoretic analysis shows only to have the reaction tubes that has mixed target nucleic acid to have amplified production, getting amplified production entrusts 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, because external double-stranded DNA is without T7 promoter sequence, can not be amplified, and this has just significantly improved the specificity of NASBA reaction; And the reaction conditions of NASBA is gentle, and shorter than the time of PCR reaction needed, therefore transcribe more faithful to template, further reduced mispairing rate.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles 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> method and test kit that detects infectious disease pathogens
<130>MP090694
<160>22
<170>PatentIn version 3.3
<210>1
<211>156
<212>DNA
<213> chlamydia psittaci endosome membranin A gene 317-472
<400>1
gccatcatgc ttgtttcgtt tgtcattgtc attatggtga ttcaggatag caccccctct 60
caagtggcac gccgtatgaa acaacaactt catcaattta gccaagaaaa cacacgctta 120
catcaagaag tagatacttt agtatctgca aacagg 156
<210>2
<211>116
<212>DNA
The main surface glycoprotein A gene 2895-3010 of <213> Pneumocystis carinii
<400>2
agcacatcta ccatcacatc taccatcaca tcaaaaataa cattgacatc aacgaggcga 60
tgcaaaccaa ccaagtgtac gacaggggat gatgcagaag acgtgaagcc aagtga 116
<210>3
<211>151
<212>DNA
<213> Leptospira gyrase B subunit gene 181-331
<400>3
ggtgaaagac aatggtcgag gaattccagt cgatattcac cccgacaaaa aaatttccac 60
aatcgaagtt gttatgacaa tccttcacgc aggtggtaaa tttgaaaatg atgcctacaa 120
agtttctgga ggtttacatg gggttggggt t 151
<210>4
<211>181
<212>DNA
The hot rickettsia outer membrane protein gene of <213>Q 703-883
<400>4
gccaattatc agaacaaatc acccttcaaa ccgcagaaaa agtaggatta aatgttgctc 60
agctcaaaaa agacatggat aatcctgcta tccaaaaaca actgcgtgat aacttccaat 120
tagctcaatc gttacagcta gcaggcaccc cgacgttcgt cattggtaat aaagcgttaa 180
c 181
<210>5
<211>236
<212>DNA
<213> brucella outer membrane protein omp2b gene 767-1002
<400>5
atcgaagaat gggctgccaa ggttcgtggc gacgtcaaca tcaccgacca gttctcggtt 60
tggttgcagg gcgcatattc gtccgctgct acgccggatc agaactacgg ccagtggggc 120
ggcgattggg ctgtctgggg tggtctgaag tatcaggcta cccagaaggc tgccttcaac 180
ctgcaggctg cgcatgacga ctggggcaag acggcagtta cggctaacgt tgctta 236
<210>6
<211>42
<212>DNA
<213> primer Chp-F
<400>6
gatgcaaggt cgcatatgag tgcttgtttc gtttgtcatt gt 42
<210>7
<211>55
<212>DNA
<213> primer Chp-R
<400>7
aattctaata cgactcacta tagggagaag gcctgtttgc agatactaaa gtatc 55
<210>8
<211>22
<212>DNA
<213> probe Chp-P
<400>8
tctcaagtcg cacgccgtat ga 22
<210>9
<211>45
<212>DNA
<213> primer Pc-F
<400>9
gatgcaaggt cgcatatgag agcacgtcta ctatcacatc tacaa 45
<210>10
<211>53
<212>DNA
<213> primer Pc-R
<400>10
aattctaata cgactcacta tagggagaag gtcacttggt ttcacgtctt ctg 53
<210>11
<211>22
<212>DNA
<213> probe Pc-P
<400>11
gcctggtttg gcaagtagcg at 22
<210>12
<211>43
<212>DNA
<213> primer Lp-F
<400>12
gatgcaaggt cgcatatgag ggtgaaagac aatggtcgag gaa 43
<210>13
<211>52
<212>DNA
<213> primer Lp-R
<400>13
aattctaata cgactcacta tagggagaag gaaccccaac cccatgtaaa cc 52
<210>14
<211>23
<212>DNA
<213> probe Lp-P
<400>14
acaatccttc acgcaggtgg taa 23
<210>15
<211>45
<212>DNA
<213> primer Qf-F
<400>15
gatgcaaggt cgcatatgag gccaattatc agaacaaatc accct 45
<210>16
<211>58
<212>DNA
<213> primer Qf-R
<400>16
aattctaata cgactcacta tagggagaag ggttaacgct ttattaccaa tgacgaac 58
<210>17
<211>27
<212>DNA
<213> probe Qf-P
<400>17
aactgcgtga taacttccaa ttagctc 27
<210>18
<211>40
<212>DNA
<213> primer Bru-F
<400>18
gatgcaaggt cgcatatgag atcgaaggat gggctgcaaa 40
<210>19
<211>54
<212>DNA
<213> primer Bru-R
<400>19
aattctaata cgactcacta tagggagaag gtaagcgacg ttggccgtaa ctgc 54
<210>20
<211>21
<212>DNA
<213> probe Bru-P
<400>20
atattcgtcc gctgctacgc c 21
<210>21
<211>20
<212>DNA
<213> capture probe
<400>21
gatgcaaggt cgcatatgag 20
<210>22
<211>236
<212>DNA
The interference sequence of the brucellar outer membrane protein omp2b of <213> gene fragment
<400>22
atcgaagatg cagctgccaa ggttcgtggc gacgtcaaca tcaccgacca gttctcggtt 60
tggttgcagg gcgcatattc gtccgctgct acgccggatc agaactacgg ccagtggggc 120
ggcgattggg ctgtctgggg tggtctgaag tatcaggcta cccagaaggc tgccttcaac 180
ctgcaggctg cgcatgacga ctggggcaag acggcagtta cggctaacgt tgctta 236
Claims (2)
1. the method that the infectious disease pathogens to being present in biological sample of non-medical diagnosis on disease or therapeutic purpose detect, comprising:
The nucleic acid fragment of (I) amplification biological sample, described nucleic acid fragment is the combination of the nucleic acid fragment as shown in SEQ ID NO.1 of chlamydia psittaci endosome membranin A gene or itself and following various nucleic acid fragments:
The nucleic acid fragment as shown in SEQ ID NO.2 of the main surface glycoprotein A gene of Pneumocystis carinii;
The nucleic acid fragment as shown in SEQ ID NO.3 of Leptospira gyrase B subunit gene;
The nucleic acid fragment as shown in SEQ ID NO.4 of the hot rickettsia outer membrane protein gene of Q;
The nucleic acid fragment as shown in SEQ ID NO.5 of brucella outer membrane protein omp2b gene;
(II) detects with probe, a kind of specific hybrid in described probe and step (I) amplified production;
Described step (I) is used the nucleic acid fragment of the 1st group of primer for increasing as shown in SEQ ID NO.1, its upstream primer nucleotide sequence as shown in SEQ ID NO.6, its downstream primer nucleotide sequence as shown in SEQ ID NO.7 or the combination of itself and following group as primer, increase:
The 2nd group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.2, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.9, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.10;
The 3rd group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.3, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.12, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.13;
The 4th group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.4, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.15, and downstream primer nucleotide sequence is as shown in SEQ ID NO.16;
The 5th group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.5, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.18, and downstream primer nucleotide sequence is as shown in SEQ ID NO.19;
Described step (I) is utilized the nucleic acid fragment of NASBA technology amplification sample, and NASBA operational conditions is 41 ℃～45 ℃ incubations 90～150 minutes;
The hybridization of probe described in step (II) and step (I) gained amplified production is carried out on solid support;
Described step (II) is used capture probe, and its nucleotide sequence is as shown in SEQ ID NO.21, and what described capture probe was direct or indirect is connected on solid support;
Use in step (II) following nucleotide sequence as detection probes:
As shown in SEQ ID NO.8, detect the nucleic acid fragment as shown in SEQ ID NO.1;
As shown in SEQ ID NO.11, detect the nucleic acid fragment as shown in SEQ ID NO.2;
As shown in SEQ ID NO.14, detect the nucleic acid fragment as shown in SEQ ID NO.3;
As shown in SEQ ID NO.17, detect the nucleic acid fragment as shown in SEQ ID NO.4;
As shown in SEQ ID NO.20, detect the nucleic acid fragment as shown in SEQ ID NO.5.
2. the test kit infectious disease pathogens that may be present in biological sample being detected, it comprises for the primer of the nucleic acid fragment as shown in SEQ ID NO.1 that increases or itself and for the primer of as shown in the SEQ ID NO.2 nucleic acid fragment of increasing, for the primer of as shown in the SEQ ID NO.3 nucleic acid fragment of increasing, for the primer of as shown in the SEQ ID NO.4 nucleic acid fragment of increasing with for a group or more combination of the primer of as shown in the SEQ ID NO.5 nucleic acid fragment of increasing;
Described primer is selected from:
The 1st group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.1, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.6, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.7;
The 2nd group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.2, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.9, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.10;
The 3rd group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.3, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.12, and its downstream primer nucleotide sequence is as shown in SEQ ID NO.13;
The 4th group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.4, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.15, and downstream primer nucleotide sequence is as shown in SEQ ID NO.16;
The 5th group of primer is used for the nucleic acid fragment increasing as shown in SEQ ID NO.5, and its upstream primer nucleotide sequence is as shown in SEQ ID NO.18, and downstream primer nucleotide sequence is as shown in SEQ ID NO.19;
Also comprise the capture probe as shown in nucleotide sequence SEQ ID NO.21;
Also comprise the detection probes that following at least one and primer pair are answered:
As shown in SEQ ID NO.8, detect the nucleic acid fragment as shown in SEQ ID NO.1;
As shown in SEQ ID NO.11, detect the nucleic acid fragment as shown in SEQ ID NO.2;
As shown in SEQ ID NO.14, detect the nucleic acid fragment as shown in SEQ ID NO.3;
As shown in SEQ ID NO.17, detect the nucleic acid fragment as shown in SEQ ID NO.4;
As shown in SEQ ID NO.20, detect the nucleic acid fragment as shown in SEQ ID NO.5.
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