CN102277452A - Kit used for detecting highly infectious disease and detection method - Google Patents
Kit used for detecting highly infectious disease and detection method Download PDFInfo
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- CN102277452A CN102277452A CN2011102379771A CN201110237977A CN102277452A CN 102277452 A CN102277452 A CN 102277452A CN 2011102379771 A CN2011102379771 A CN 2011102379771A CN 201110237977 A CN201110237977 A CN 201110237977A CN 102277452 A CN102277452 A CN 102277452A
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Abstract
The invention relates to a kit used for detecting high infectious disease and also provides a highly infectious disease virus detection method adopting the kit. The kit comprises an amplification primer and a detection probe which are designed according to a gene conserved region of a highly infectious disease virus, nucleic acid segment in a sample to be detected is extracted and amplified by virtue of an amplification primer, then the nucleic acid segment is specifically combined with the amplification primer by utilizing the detection probe, and whether the sample contains the highly infectious disease virus to be detected is detected. The detection method adopting the kit has short time, high sensitivity and strong practicability.
Description
[technical field]
The present invention relates to biology field, relate in particular to a kind of deadly infectious disease germ detection kit and detection method.
[background technology]
Deadly infectious disease virus can cause the human infectious disease, and higher mortality ratio is arranged, and comprises Hantaan virus, west nile virus, yellow fever virus, Crimea-congo fever virus, lassa virus, Rift Valley fever virus, Marburg virus, Ebola virus.Detection to deadly infectious disease virus is vital for human infectious disease's diagnosis and control.
The detection of traditional deadly infectious disease virus mainly is the method that adopts microbial culture, and this method detected result is more accurate, but expends time in length, complex operation step; In addition, can also adopt the real-time fluorescence PCR method to detect, this method high specificity, but once experiment can only detect a kind of cause of disease target compound, detects a large amount of pathogenic bacterias simultaneously and then can not deal with well.But these two kinds of methods often need to expend the long time when the multiple deadly infectious disease of detection is viral, the best moment that this has also incured loss through delay diagnosis and has treated.
[summary of the invention]
Based on this, be necessary to provide a kind of deadly infectious disease method for detecting virus that can detect the deadly infectious disease virus detection kit of multiple deadly infectious disease virus at short notice and adopt above-mentioned deadly infectious disease virus detection kit.
A kind of deadly infectious disease virus detection kit comprises being selected from least a in following 8 groups of primers:
The 1st group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.1 of Hantaan virus;
The 2nd group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.2 of west nile virus;
The 3rd group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.3 of yellow fever virus;
The 4th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.4 of Crimea-congo fever virus;
The 5th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.5 of lassa virus;
The 6th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.6 of Marburg virus;
The 7th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.7 of Rift Valley fever virus;
The 8th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.8 of Ebola virus.
Preferably, described the 1st group of primer: the upstream primer sequence is shown in SEQ ID NO.9, and the downstream primer sequence is shown in SEQ ID NO.17;
Described the 2nd group of primer: the upstream primer sequence is shown in SEQ ID NO.10, and the downstream primer sequence is shown in SEQ ID NO.18;
Described the 3rd group of primer: the upstream primer sequence is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQID NO.19;
Described the 4th group of primer: the upstream primer sequence is shown in SEQ ID NO.12, and the downstream primer sequence is shown in SEQ ID NO.20;
Described the 5th group of primer: the upstream primer sequence is shown in SEQ ID NO.13, and the downstream primer sequence is shown in SEQ ID NO.21;
Described the 6th group of primer: the upstream primer sequence is shown in SEQ ID NO.14, and the downstream primer sequence is shown in SEQ ID NO.22;
Described the 7th group of primer: the upstream primer sequence is shown in SEQ ID NO.15, and the downstream primer sequence is shown in SEQ ID NO.23;
Described the 8th group of primer: the upstream primer sequence is shown in SEQ ID NO.16, and the downstream primer sequence is shown in SEQ ID NO.24.
Preferably, also comprise detection probes, described detection probes is that following 8 nucleic acid fragments corresponding with described 8 groups of primers are at least a:
The 1st detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.1, and sequence is shown in SEQID NO.25;
The 2nd detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.2, and sequence is shown in SEQID NO.26;
The 3rd detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.3, and sequence is shown in SEQID NO.27;
The 4th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.3, and sequence is shown in SEQID NO.28;
The 5th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.4, and sequence is shown in SEQID NO.29;
The 6th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.5, and sequence is shown in SEQID NO.30;
The 7th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.6, and sequence is shown in SEQID NO.31;
The 8th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.7, and sequence is shown in SEQID NO.32.
Preferably, also comprise solid phase carrier, described solid phase carrier comprises matrix and the adhesion layer that is fixed on the described matrix, and described detection probes is fixed on the described adhesion layer.
Preferably, 5 ' of described detection probes end is connected with 15 thymus pyrimidines.
Preferably, also comprise the positive quality control probe and with described positive quality control probe complementary positive detection probe;
Described positive quality control probe sequence is shown in SEQ ID NO.33, and the sequence of described positive detection probe is shown in SEQ ID NO.34.
A kind of detection method of deadly infectious disease virus comprises the steps:
Nucleic acid in step 1, the extraction testing sample;
Step 2, the nucleic acid of amplification in the testing sample obtain nucleic acid fragment, and described nucleic acid fragment is at least a in the following nucleic acid fragment:
The nucleic acid fragment shown in SEQ ID NO.1 of Hantaan virus;
The nucleic acid fragment shown in SEQ ID NO.2 of west nile virus;
The nucleic acid fragment shown in SEQ ID NO.3 of yellow fever virus;
The nucleic acid fragment shown in SEQ ID NO.4 of Crimea-congo fever virus;
The nucleic acid fragment shown in SEQ ID NO.5 of lassa virus;
The nucleic acid fragment shown in SEQ ID NO.6 of Marburg virus;
The nucleic acid fragment shown in SEQ ID NO.7 of Rift Valley fever virus;
The nucleic acid fragment shown in SEQ ID NO.8 of Ebola virus;
Step 3, detect a kind of specific hybrid in described detection probes and the step 2 amplified production with detection probes.
Preferably, step 2 uses at least one group of following 8 groups of oligonucleotide fragments to increase as primer:
Described the 1st group of primer: the upstream primer sequence is shown in SEQ ID NO.9, and the downstream primer sequence is shown in SEQID NO.17;
Described the 2nd group of primer: the upstream primer sequence is shown in SEQ ID NO.10, and the downstream primer sequence is shown in SEQ ID NO.18;
Described the 3rd group of primer: the upstream primer sequence is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQID NO.19;
Described the 4th group of primer: the upstream primer sequence is shown in SEQ ID NO.12, and the downstream primer sequence is shown in SEQ ID NO.20;
Described the 5th group of primer: the upstream primer sequence is shown in SEQ ID NO.13, and the downstream primer sequence is shown in SEQ ID NO.21;
Described the 6th group of primer: the upstream primer sequence is shown in SEQ ID NO.14, and the downstream primer sequence is shown in SEQ ID NO.22;
Described the 7th group of primer: the upstream primer sequence is shown in SEQ ID NO.15, and the downstream primer sequence is shown in SEQ ID NO.23;
Described the 8th group of primer: the upstream primer sequence is shown in SEQ ID NO.16, and the downstream primer sequence is shown in SEQ ID NO.24.
Preferably, detection probes described in the step 3 is at least one that has in 8 nucleic acid fragments of following sequence:
The 1st detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.1, and sequence is shown in SEQID NO.25;
The 2nd detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.2, and sequence is shown in SEQID NO.26;
The 3rd detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.3, and sequence is shown in SEQID NO.27;
The 4th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.3, and sequence is shown in SEQID NO.28;
The 5th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.4, and sequence is shown in SEQID NO.29;
The 6th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.5, and sequence is shown in SEQID NO.30;
The 7th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.6, and sequence is shown in SEQID NO.31;
The 8th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.7, and sequence is shown in SEQID NO.32.
Preferably, the hybridization of detection probes described in the step 3 and described amplified production is carried out on solid phase carrier;
Described solid phase carrier comprises matrix and the adhesion layer that is fixed on the described matrix, and described detection probes is fixed on the described adhesion layer.
Gene conserved regions design amplimer and detection probes according to deadly infectious disease virus, extract the nucleic acid fragment in the testing sample and increase by amplimer, using detection probes and amplified production to carry out specificity at last combines, whether contain deadly infectious disease virus to be detected in the test sample, adopt the detection method of this test kit short and highly sensitive detection time, have very strong practicality.
[description of drawings]
Fig. 1 is the schema of detection method of the deadly infectious disease virus of an embodiment;
Fig. 2 is the electrophorogram of amplified production after the Hantaan virus sample nucleic acid extraction;
Fig. 3 is the detection dot matrix synoptic diagram of the deadly infectious disease virus of an embodiment;
Fig. 4 detects the detected result synoptic diagram that dot matrix detects Hantaan virus for Fig. 3 shows.
[embodiment]
Mainly in conjunction with the accompanying drawings and embodiments deadly infectious disease virus detection kit and detection method are further explained explanation below.
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, detection probes
By 8 kinds of common deadly infectious disease virus gene sequences are analyzed, selecting conservative relatively sequence is the target extension increasing sequence, and according to this target extension increasing sequence be designed for this target extension increasing sequence of amplification amplimer and with this target extension increasing sequence specificity bonded detection probes.
8 target extension increasing sequences and corresponding 8 groups of amplimers and 8 detection probes thereof are as follows:
The 1st target extension increasing sequence is the nucleic acid fragment shown in SEQ ID NO.1 of Hantaan virus, and corresponding upstream primer sequence is shown in SEQ ID NO.9, and the downstream primer sequence is shown in SEQ ID NO.17, and the detection probes sequence is shown in SEQ ID NO.25.
The 2nd target extension increasing sequence is the nucleic acid fragment shown in SEQ ID NO.2 of west nile virus, and corresponding upstream primer sequence is shown in SEQ ID NO.10, and the downstream primer sequence is shown in SEQ ID NO.18, and the detection probes sequence is shown in SEQ ID NO.26.
The 3rd target extension increasing sequence is the nucleic acid fragment shown in SEQ ID NO.3 of yellow fever virus, and corresponding upstream primer sequence is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQ ID NO.19, and the detection probes sequence is shown in SEQ ID NO.27.
The 4th target extension increasing sequence is the nucleic acid fragment shown in SEQ ID NO.4 of Crimea-congo fever virus, corresponding upstream primer sequence is shown in SEQ ID NO.12, the downstream primer sequence is shown in SEQ IDNO.20, and the detection probes sequence is shown in SEQ ID NO.28.
The 5th target extension increasing sequence is the nucleic acid fragment shown in SEQ ID NO.5 of lassa virus, and corresponding upstream primer sequence is shown in SEQ ID NO.13, and the downstream primer sequence is shown in SEQ ID NO.21, and the detection probes sequence is shown in SEQ ID NO.29.
The 6th target extension increasing sequence is the nucleic acid fragment shown in SEQ ID NO.6 of Marburg virus, and corresponding upstream primer sequence is shown in SEQ ID NO.14, and the downstream primer sequence is shown in SEQ ID NO.22, and the detection probes sequence is shown in SEQ ID NO.30.
The 7th target extension increasing sequence is the nucleic acid fragment shown in SEQ ID NO.7 of Rift Valley fever virus, and corresponding upstream primer sequence is shown in SEQ ID NO.15, and the downstream primer sequence is shown in SEQ ID NO.23, and the detection probes sequence is shown in SEQ ID NO.31.
The 8th target extension increasing sequence is the nucleic acid fragment shown in SEQ ID NO.8 of Ebola virus, and corresponding upstream primer sequence is shown in SEQ ID NO.16, and the downstream primer sequence is shown in SEQ ID NO.24, and the detection probes sequence is shown in SEQ ID NO.32.
8 downstream primer 5 ' ends are marked with vitamin H, are used for follow-up color reaction.
8 detection probes 5 ' ends are connected with 15 thymus pyrimidines (T), make detection probes better to extend, and are beneficial to the hybridization of detection probes and amplified production.
Above-mentioned primer and probe are synthetic by Shanghai bio-engineering corporation.
Embodiment 2
Deadly infectious disease virus detection kit and detection method
Detection kit comprises above-mentioned 8 groups of primers and 8 probes.
In other embodiment, detection kit can comprise in above-mentioned 8 groups of primers and 8 probes any one or a few.Primer that contains and probe combinations the more, the corresponding viral species that can detect is the more.
Detection kit in the present embodiment comprises above-mentioned 8 groups of primers and 8 probes.
In a preferred embodiment, detection kit also comprise the positive quality control probe and with positive quality control probe complementary positive detection probe; The sequence of positive quality control probe is shown in SEQ ID NO.33, and the sequence of positive detection probe is shown in SEQ ID NO.34.
The detection method of deadly infectious disease virus as shown in Figure 1 comprises the steps:
Nucleic acid in S10, the extraction testing sample
Nucleic acid extraction adopts the pillar viral RNA OUT of sky, Beijing bounties Gene Tech. Company Limited to extract test kit and extracts (production code member 71001-50).
Using method is specifically as follows:
1, for the liquid viral sample: in the 1.5mL centrifuge tube, add 0.1～0.2mL liquid viral sample.If virus needs enrichment, can be with 1.5mL liquid at 4 ℃ 24,000g frozen centrifugation 60min moves and abandons 0.2mL remaining behind the 1.3mL and be used for next step processing.
For the swab sample: a swab is put into centrifuge tube, add the physiological saline that 1mL provides for oneself, abundant vibration half a minute on the vibrator, transferase 10 .2mL is used for next step processing in the 1.5mL plastic centrifuge tube then.
2, add in the 0.2mL sample that the 0.6mL lysate obtains to the first step, the room temperature placement is 10 minutes behind the 30 seconds mixings of vibrate.Wherein, lysate may produce precipitation after 4 ℃ of placements, must be placed on before using to take after 65 ℃ of water-baths also fully shake up the thorough dissolving of precipitation again.
3,12, centrifugal 30 seconds of 000rpm transfers to 500 μ L solution in the centrifugal adsorption column, and room temperature was placed 2 minutes.
4,12, centrifugal 1 minute of 000rpm room temperature is abandoned and is contained the collection tube that penetrates liquid, and centrifugal adsorption column is put in the new collection tube.Penetrate liquid and be the liquid that is thrown to after centrifugal in the collection tube.
5, after the step 3, all transfer in the centrifugal adsorption column after centrifugal surplus solution in 2 is of short duration, room temperature was placed 2 minutes.
6,12, centrifugal 1 minute of 000rpm room temperature is abandoned and is contained the collection tube that penetrates liquid, and centrifugal adsorption column is put in the new collection tube.
7, add the general post liquid of washing of 0.7mL in centrifugal adsorption column, 12, centrifugal 1 minute of 000rpm room temperature is abandoned and is contained the collection tube that penetrates liquid, centrifugal adsorption column is put in the 1.5mL centrifuge tube of a RNase-free who provides for oneself.
8,12, centrifugal 1 minute of 000rpm room temperature is abandoned and is contained the centrifuge tube that penetrates liquid.
9, centrifugal adsorption column is nested in the 1.5mL centrifuge tube of a new RNase-free who provides for oneself, adds the RNA elutriant of 30～100 μ L at the middle part of centrifugal adsorption column filter membrane, room temperature was placed 2 minutes then.
10,12, centrifugal 1 minute of 000rpm room temperature, solution is the RNA solution of viral sample in the collection tube, is designated as template ribonucleic acid.
Nucleic acid in S20, the amplification testing sample obtains nucleic acid fragment
Adopting conventional amplification system, is example with the amplification system of 50 μ L, and amplification system is as follows:
Sterilized water complements to 50 μ L and gets final product.
Place in the PCR reaction instrument, reaction conditions is:
Template ribonucleic acid elder generation reverse transcription under the catalysis of AMV ThermoScript II obtains corresponding cDNA sequence, and PCR obtains the purpose fragment again.
Negative control adopts 5 μ L aqua sterilisas to replace the template ribonucleic acid of test group.
After finishing, amplification carries out electrophoresis, and whether successful to judge amplification.
In the above-mentioned experiment, many group primers can be added simultaneously,, also a kind of virus can be only detected to detect multiple virus.
Only to detect Hantaan virus is example, the upstream primer sequence shown in SEQ ID NO.9, the downstream primer sequence shown in SEQ ID NO.17, other conditions and operation steps such as above-mentioned, amplification obtains nucleic acid fragment shown in SEQ ID NO.1.
Carry out electrophoresis after amplification is finished, obtain electrophorogram as shown in Figure 2, Marker adopts dna molecular amount standard DL2000, as can be seen from Figure, occurs the purpose band in the running gel, and amplified reaction carries out smoothly.
S30, detect in the judgement sample whether have deadly infectious disease virus with detection probes
Comprise the steps:
S32, detection probes is fixed on the solid phase carrier.
Can select pan coating that the solid phase carrier of 96 microwell plates of one or more mixtures in poly-l-lysine layer, Streptavidin layer, gsh layer, pentanedial decoration layer or complexon I-nickel ion layer as probe stationary arranged, probe is fixed on the described bag tegillum with the form of lattice array; Wherein, the fixed form of probe is for Covalent Immobilization or orientation are fixing at random.
Present embodiment selects pan coating that the solid phase carrier of 96 microwell plates of poly-l-lysine layer as probe stationary arranged, and stylus orientation is fixed on the bag tegillum.
Concrete steps are: the synthetic probe molecule is dissolved in the distilled water, and ultimate density is decided to be 20 μ M, gets the above-mentioned probe solution of 5 μ L and 5 μ L, 2 * crystalline substance
(CapitalBio Corporation, catalog number: 440010), and with behind the pipettor thorough mixing, the gained mixed solution can carry out the point sample of biochip to gene chip sampling liquid, and point sample generally adopts the form of dot matrix.Behind the point sample detection probes is fixed on the adhesion layer, detection probes 5 ' end is connected with 15 thymus pyrimidines (T) simultaneously, makes detection probes better to extend, and is beneficial to the hybridization of detection probes and amplified production.
In a preferred embodiment, dot matrix comprises detection probes point and positive quality control probe points, and the amplified production that the detection probes point is used for obtaining with S20 is hybridized, and positive quality control point is used to detect the success or not of hybrid experiment.Detection probes point and positive quality control probe points are distributed on the chip with the form of lattice array, each dot matrix comprises 4 positive quality control probe points and several detection probes points, the positive quality control probe that 4 positive quality control probe points are adopted is the oligonucleotide sequence of one section synthetic, contain the oligonucleotide positive detection probe that is all synthetic with one section of positive quality control probe complementary in the hybridization reaction solution, the positive quality control probe sequence is shown in SEQ IDNO.33, and the sequence of positive detection probe is shown in SEQ ID NO.34.Whether the positive quality control probe combines with the positive detection probe specificity during hybridization, correct in order to the check crossover process through follow-up color reaction again.
Detecting object point can be 1, also can mostly be 8 most for a plurality of.
Can put a plurality of holes at 96 microwell plate mid points and do detection, promptly many group dot matrix, every group of dot matrix arrangement is identical, in order to compare.If each hole reaction result is all identical, then ecbatic is accurate, if there is hole inequality the experiment of then need reforming to occur.Concrete can select 2 dot matrix, 4 dot matrix etc.
The detection dot matrix of an embodiment as shown in Figure 3, specifically make up as follows:
In actually operating be, can select to detect virus, also can select to detect with the dot matrix of other modes with above-mentioned dot matrix.
37 ℃ of hydrations of excellent 96 microwell plates of point are spent the night, and distilled water cleans 2 times, and 96 microwell plates are put into borate solution (1.0g NaBH
4+ 300mL PBS+100mL dehydrated alcohol) hatch 5min in, distilled water cleans 2 times, behind the air drying, promptly can be used for detecting.
S34, the amplified production that uses excellent 96 microwell plates of point and S20 to obtain carry out hybridization.
The amplified production that S20 obtains takes out and places 5min on ice immediately after being heated to 99 ℃ of 5min on the PCR instrument, obtains the sex change liquid of amplified production.
Every hole adds sex change liquid 20 μ L respectively in excellent four holes of 96 microwell plate mid points that S34 obtains, and hybridization reaction solution 200 μ L stick and put into the hybridization case behind the glued membrane, and 45 ℃ of hybridization 2h make abundant reaction.
Described hybridization liquid formula is: 98.9g dextran sulfate sodium (Dextran Sulfate Sodium), 1.99g toxilic acid (Maleic acid), 2.1g NaOH, 0.87g NaCl, 6.5g boric acid (Boric acid), 10g BSA (Bovine Serum Albumin, bovine serum albumin), be dissolved in the 1000mL distilled water, contain positive probe in the described hybridization reaction solution.
Hybridization is torn glued membrane after finishing, and reaction solution is poured out.In each reacting hole, add hybridization washings 200 μ L, shook microwell plate back and forth 20 seconds (do not overflow with liquid in the hole and exceed), firmly outwell the liquid in the hole then, repeat this action 3 times with hand.After last is outwelled all over liquid 96 microwell plates are placed and pat on the thieving paper for several times, to remove residual liquid.
Hybridization washings prescription is: 0.6g NaCl, 0.7g NaH
2PO
42H
2O, 8.9g SDS (Sodium Dodecyl Sulfonate, sodium laurylsulfonate), 100mL 20 * SSC is dissolved in the 1000mL distilled water.The prescription of described 20 * SSC is: 175.3g NaCl, 88.2g Na
3C
6H
5O
72H
2O (Trisodium Citrate, Sodium Citrate) is dissolved in 1000mL DEPC (Diethyl Pyrocarbonate, the diethylpyrocarbonate) water, and HCl regulates pH to 7.0, autoclaving, after the cooling promptly.
After above-mentioned cleaning finishes, in each reacting hole, add 200 μ L and be marked with the solution of streptavidin of alkaline phosphatase (Streptavidin-Alkaline phosphatase Strep-Ap), reacts 20min under room temperature.The Streptavidin that is marked with alkaline phosphatase can carry out affinity interaction with vitamin H on the pcr amplification product and the vitamin H on the positive probe, makes the alkaline phosphatase can be attached on 96 microwell plates.
Described solution of streptavidin is the mixed solution of Strep-Ap solution and its diluent, and the prescription of diluent is: 3.11g NaOH, and 150mL 0.1%Tween-20,10g BSA is dissolved in the 1000mL distilled water.Strep-Ap solution mixes by 1: 1000 volume ratio with its diluent before using.
After above-mentioned reaction finishes, in each reacting hole, add 200 μ L colour generation mixed solutions, lucifuge reaction 10min under room temperature.The colour generation mixed solution is for being the chromogen agent and being chromogen dilution agent liquid mixing gained.The composition that is the chromogen agent is NBT/BCIP (NBT:Tetranitro Blue Tetrazolium Chloride, a NBT; BCIP:5-Bromo-4-Chloro-3-Indolyl Phosphate, 5-bromo-4-chloro-3-indyl-phosphoric acid salt), can carry out oxidation and reduction reaction with alkaline phosphatase, reaction can produce macroscopic bluish voilet sedimentable matter simultaneously, concrete prescription is: 2.4g NBT, 0.5g BCIP is dissolved in 100mL DMSO (dimethyl sulfoxide (DMSO)) aqueous solution.Be chromogen dilution agent liquid and can guarantee that alkaline phosphatase activity is normal, be chromogen dilution agent liquid formula and be: 1.23g MgCl
26H
2O, 1.32g NaCl, 0.8g HCl is dissolved in the 1000mL distilled water.Be the chromogen agent before the use and be chromogen dilution agent liquid and mix by 1: 50 volume ratio.
S36, interpretation as a result
Colour generation appears in four positive quality control point, illustrates that promptly the hybridization process is correct, judges whether there are in 8 kinds of Respiroviruses one or more in the testing sample by detecting the object point change in color again.
To detect Hantaan virus is example, carries out 4 group reactions simultaneously, adopts dot matrix as shown in Figure 3 to detect.
Detected result synoptic diagram as shown in Figure 4 is that the amplified production of Hantaan virus nucleic acid fragment adopts Fig. 3 to show the result schematic diagram that detects the dot matrix detection, carries out four group reactions simultaneously.
As can be seen from Figure, colour developing all appears in the A1 of four groups of dot matrix, A5, E1 and E5, illustrates that hybridization successfully carries out; Colour developing all appears in B4 simultaneously, can judge and contain Hantaan virus in the testing sample.
Gene conserved regions design amplimer and probe according to deadly infectious disease virus, extract the nucleic acid of testing sample and increase by amplimer, use probe and amplified production to carry out specific hybrid at last, whether contain Respirovirus to be detected in the test sample, this detection method detect the flux height, fast, sensitivity and high specificity.
The probe molecule point of a plurality of detection targets is put on 96 microwell plates, detection flux height by positive quality control point is set, and adds in hybridization reaction solution and the positive probe of positive quality control probe complementary, can judge easily whether hybridization is correct, thereby reduce experimental error.
Simultaneously, adopt the multiplex amplification technology, disposablely can carry out specific amplification, thereby further accelerated the speed that detects at the nucleic acid of above-mentioned 8 kinds of deadly infectious disease viruses.
SEQUENCE?LISTING
＜110〉Shenzhen International Travel Health Care Center, Shenzhen Bo Ermei bio tech ltd
＜120〉deadly infectious disease virus detection kit and detection method
<160>34
<170>PatentIn?version?3.3
<210>1
<211>100
<212>DNA
＜213〉Hantaan virus
<400>1
gaaaaaaact?gggatgagtg?acttgacaga?cattcaggag?gatataaccc?gccatgaaca 60
acagcttgtt?gttgccagac?aaaagctcaa?ggatgcagaa 100
<210>2
<211>104
<212>DNA
＜213〉west nile virus
<400>2
aaagcgaatt?tcagtgtgat?ttttgaccgg?ctagagaccc?taatattact?aagggctttc 60
accgaagagg?gagcaattgt?tggcgaaatc?tcaccattgc?cttc 104
<210>3
<211>107
<212>DNA
＜213〉yellow fever virus
<400>3
gttagaggag?accctccagg?gaacaaatag?tgggaccata?ttgacgccag?ggaaagaccg 60
gagtggttct?ctgcttttcc?tccagaggtc?tgtgagcaca?gtttgct 107
<210>4
<211>359
<212>DNA
＜213〉crimean-Congo hemorrhagic fever virus
<400>4
aaaatacagg?aactattaaa?tcttgggatg?agagttatac?tgagctgaaa?gttgaagttc 60
ccaaaataga?acaacttttc?aactaccagc?aggctgctct?caagtggagg?aaagacatag 120
gtttccgtgt?caatgcaaac?acggcagctt?taagtaacaa?agtcctagca?gaatacaaag 180
ttcctggcga?aattgtaatg?tctgttaaag?agatgttgtc?agatatgatt?agaaggagga 240
tgagctcttt?gccgatgatt?ctttccagca?gaacaggatt?tacatgcatc?ctgccgtgct 300
tacagctggt?agaatcagtg?aaatgggagt?ctgctttggg?acaatccctg?tggccaatc 359
<210>5
<211>130
<212>DNA
＜213〉lassa virus
<400>5
caaggggctc?gggctgggag?agatggagtg?gtgagagttt?gggatgtgaa?aaatgcagag 60
ttgctcaata?atcagttcgg?gaccatgcca?agtctgacac?tggcatgtct?gacaaaacag 120
gggcaggttg 130
<210>6
<211>77
<212>DNA
＜213〉Marburg virus
<400>6
caattccacc?ttcagaaaac?tgaaatcaca?cacagtcaga?cactagccgt?cctcagccag 60
aaacgagaaa?aattagc 77
<210>7
<211>155
<212>DNA
＜213〉Rift Valley fever virus
<400>7
tttccacaat?atttaagtgt?caattatttg?catcgcctta?cagtcagtag?tagaccatgt 60
gaattccctg?catcaatacc?agcttataga?acaacaaatt?atcactttga?cactagccct 120
attaatcgca?tattaacaga?aaagtatggt?gatga 155
<210>8
<211>580
<212>DNA
＜213〉Ebola virus
<400>8
gaatgggcag?aaaattgcta?caatctggag?atcaaaaagt?cagacggaag?tgaatgcctc 60
cctctccctc?ccgacggtgt?acgaggattc?cctagatgtc?gctatgtcca?caaagttcaa 120
ggaacaggtc?cttgtcctgg?tgacttagct?ttccataaaa?atggggcttt?tttcttgtat 180
gatagattgg?cctcaactgt?catctaccga?gggacaactt?ttgctgaagg?tgtcgtagct 240
tttttaattc?tgtcagagcc?caagaagcat?ttttggaagg?ctacaccagc?tcatgaaccg 300
gtgaacacaa?cagatgattc?cacaagctac?tacatgaccc?tgacactcag?ctacgagatg 360
tcaaattttg?ggggcaatga?aagcaacacc?ctttttaagg?tagacaacca?caeatatgtg 420
caactagatc?gtccacacac?tccgcagttc?cttgttcagc?tcaatgaaac?acttcgaaga 480
aataatcgcc?ttagcaacag?tacagggaga?ttgacttgga?cattggatcc?taaaattgaa 540
ceagatgttg?gtgagtgggc?cttctgggaa?actaaaaaaa 580
<210>9
<211>22
<212>DNA
＜213〉artificial sequence
<400>9
aaaaactggg?atgagtgact?tg 22
<210>10
<211>23
<212>DNA
＜213〉artificial sequence
<400>10
ttctgcatcc?ttgagctttt?gtc 23
<210>11
<211>25
<212>DNA
＜213〉artificial sequence
<400>11
cctgtgtgag?ctgacaaact?tagta 25
<210>12
<211>25
<212>DNA
＜213〉artificial sequence
<400>12
cttagacatc?gagatcttcg?tgcca 25
<210>13
<211>19
<212>DNA
＜213〉artificial sequence
<400>13
gggaacaaat?agtgggacc 19
<210>14
<211>18
<212>DNA
＜213〉artificial sequence
<400>14
gacctctgga?ggaaaagc 18
<210>15
<211>18
<212>DNA
＜213〉artificial sequence
<400>15
taggtttccg?tgtcaatg 18
<210>16
<211>18
<212>DNA
＜213〉artificial sequence
<400>16
gtcccaaagc?agactccc 18
<210>17
<211>25
<212>DNA
＜213〉artificial sequence
<400>17
ggggctcggg?ctgggagaga?tggag 25
<210>18
<211>25
<212>DNA
＜213〉artificial sequence
<400>18
ctgcccctgt?tttgtcagac?atgcc 25
<210>19
<211>22
<212>DNA
＜213〉artificial sequence
<400>19
caattccacc?ttcagaaaac?tg 22
<210>20
<211>23
<212>DNA
＜213〉artificial sequence
<400>20
gctaattttt?ctcgtttctg?gct 23
<210>21
<211>20
<212>DNA
＜213〉artificial sequence
<400>21
attcctgaga?cacatggcat 20
<210>22
<211>20
<212>DNA
＜213〉artificial sequence
<400>22
cacttccttg?catcatctga 20
<210>23
<211>24
<212>DNA
＜213〉artificial sequence
<400>23
aatgggctga?aaattgctac?aatc 24
<210>24
<211>27
<212>DNA
＜213〉artificial sequence
<400>24
tttttttagt?ttcccagaag?gcccact 27
<210>25
<211>25
<212>DNA
＜213〉artificial sequence
<400>25
ataacccgcc?atgaacaaca?gcttg 25
<210>26
<211>20
<212>DNA
＜213〉artificial sequence
<400>26
cagtgcgagc?tgtttcttgg 20
<210>27
<211>21
<212>DNA
＜213〉artificial sequence
<400>27
tattgacgcc?agggaaagac?c 21
<210>28
<211>19
<212>DNA
＜213〉artificial sequence
<400>28
ttgccgatga?ttctttcca 19
<210>29
<211>33
<212>DNA
＜213〉artificial sequence
<400>29
aatgcagagt?tgctcaataa?tcagttcggg?acc 33
<210>30
<211>27
<212>DNA
＜213〉artificial sequence
<400>30
cacacacagt?cagacactag?ccgtcct 27
<210>31
<211>18
<212>DNA
＜213〉artificial sequence
<400>31
gccccttaca?ttgcttcc 18
<210>32
<211>25
<212>DNA
＜213〉artificial sequence
<400>32
aaaatggggc?ttttttcttg?tatga 25
<210>33
<211>23
<212>DNA
＜213〉artificial sequence
<400>33
atcgttcgtg?acatcaagga?gaa 23
<210>34
<211>23
<212>DNA
＜213〉artificial sequence
<400>34
tagcaagcac?tgtagttcct?ctt 23
Claims (10)
1. a deadly infectious disease virus detection kit is characterized in that, comprises being selected from least a in following 8 groups of primers:
The 1st group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.1 of Hantaan virus;
The 2nd group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.2 of west nile virus;
The 3rd group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.3 of yellow fever virus;
The 4th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.4 of Crimea-congo fever virus;
The 5th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.5 of lassa virus;
The 6th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.6 of Marburg virus;
The 7th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.7 of Rift Valley fever virus;
The 8th group of primer be used to increase nucleic acid fragment shown in SEQ ID NO.8 of Ebola virus.
2. deadly infectious disease virus detection kit as claimed in claim 1 is characterized in that,
Described the 1st group of primer: the upstream primer sequence is shown in SEQ ID NO.9, and the downstream primer sequence is shown in SEQID NO.17;
Described the 2nd group of primer: the upstream primer sequence is shown in SEQ ID NO.10, and the downstream primer sequence is shown in SEQ ID NO.18;
Described the 3rd group of primer: the upstream primer sequence is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQID NO.19;
Described the 4th group of primer: the upstream primer sequence is shown in SEQ ID NO.12, and the downstream primer sequence is shown in SEQ ID NO.20;
Described the 5th group of primer: the upstream primer sequence is shown in SEQ ID NO.13, and the downstream primer sequence is shown in SEQ ID NO.21;
Described the 6th group of primer: the upstream primer sequence is shown in SEQ ID NO.14, and the downstream primer sequence is shown in SEQ ID NO.22;
Described the 7th group of primer: the upstream primer sequence is shown in SEQ ID NO.15, and the downstream primer sequence is shown in SEQ ID NO.23;
Described the 8th group of primer: the upstream primer sequence is shown in SEQ ID NO.16, and the downstream primer sequence is shown in SEQ ID NO.24.
3. deadly infectious disease virus detection kit as claimed in claim 1 or 2 is characterized in that, also comprises detection probes, and described detection probes is that following 8 nucleic acid fragments corresponding with described 8 groups of primers are at least a:
The 1st detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.1, and sequence is shown in SEQID NO.25;
The 2nd detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.2, and sequence is shown in SEQID NO.26;
The 3rd detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.3, and sequence is shown in SEQID NO.27;
The 4th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.3, and sequence is shown in SEQID NO.28;
The 5th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.4, and sequence is shown in SEQID NO.29;
The 6th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.5, and sequence is shown in SEQID NO.30;
The 7th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.6, and sequence is shown in SEQID NO.31;
The 8th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.7, and sequence is shown in SEQID NO.32.
4. deadly infectious disease virus detection kit as claimed in claim 3 is characterized in that, also comprises solid phase carrier, and described solid phase carrier comprises matrix and the adhesion layer that is fixed on the described matrix, and described detection probes is fixed on the described adhesion layer.
5. deadly infectious disease virus detection kit as claimed in claim 3 is characterized in that, 5 ' end of described detection probes is connected with 15 thymus pyrimidines.
6. deadly infectious disease virus detection kit as claimed in claim 1 is characterized in that, also comprise the positive quality control probe and with described positive quality control probe complementary positive detection probe;
Described positive quality control probe sequence is shown in SEQ ID NO.33, and the sequence of described positive detection probe is shown in SEQ ID NO.34.
7. the detection method of a deadly infectious disease virus is characterized in that, comprises the steps:
Nucleic acid in step 1, the extraction testing sample;
Step 2, the nucleic acid of amplification in the testing sample obtain nucleic acid fragment, and described nucleic acid fragment is at least a in the following nucleic acid fragment:
The nucleic acid fragment shown in SEQ ID NO.1 of Hantaan virus;
The nucleic acid fragment shown in SEQ ID NO.2 of west nile virus;
The nucleic acid fragment shown in SEQ ID NO.3 of yellow fever virus;
The nucleic acid fragment shown in SEQ ID NO.4 of Crimea-congo fever virus;
The nucleic acid fragment shown in SEQ ID NO.5 of lassa virus;
The nucleic acid fragment shown in SEQ ID NO.6 of Marburg virus;
The nucleic acid fragment shown in SEQ ID NO.7 of Rift Valley fever virus;
The nucleic acid fragment shown in SEQ ID NO.8 of Ebola virus;
Step 3, detect a kind of specific hybrid in described detection probes and the step 2 amplified production with detection probes.
8. the detection method of deadly infectious disease virus as claimed in claim 7 is characterized in that, step 2 uses at least one group of following 8 groups of oligonucleotide fragments to increase as primer:
Described the 1st group of primer: the upstream primer sequence is shown in SEQ ID NO.9, and the downstream primer sequence is shown in SEQID NO.17;
Described the 2nd group of primer: the upstream primer sequence is shown in SEQ ID NO.10, and the downstream primer sequence is shown in SEQ ID NO.18;
Described the 3rd group of primer: the upstream primer sequence is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQID NO.19;
Described the 4th group of primer: the upstream primer sequence is shown in SEQ ID NO.12, and the downstream primer sequence is shown in SEQ ID NO.20;
Described the 5th group of primer: the upstream primer sequence is shown in SEQ ID NO.13, and the downstream primer sequence is shown in SEQ ID NO.21;
Described the 6th group of primer: the upstream primer sequence is shown in SEQ ID NO.14, and the downstream primer sequence is shown in SEQ ID NO.22;
Described the 7th group of primer: the upstream primer sequence is shown in SEQ ID NO.15, and the downstream primer sequence is shown in SEQ ID NO.23;
Described the 8th group of primer: the upstream primer sequence is shown in SEQ ID NO.16, and the downstream primer sequence is shown in SEQ ID NO.24.
9. the detection method of deadly infectious disease virus as claimed in claim 7 is characterized in that, detection probes described in the step 3 is at least one that has in 8 nucleic acid fragments of following sequence:
The 1st detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.1, and sequence is shown in SEQID NO.25;
The 2nd detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.2, and sequence is shown in SEQID NO.26;
The 3rd detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.3, and sequence is shown in SEQID NO.27;
The 4th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.3, and sequence is shown in SEQID NO.28;
The 5th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.4, and sequence is shown in SEQID NO.29;
The 6th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.5, and sequence is shown in SEQID NO.30;
The 7th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.6, and sequence is shown in SEQID NO.31;
The 8th detection probes combines with nucleic acid fragment specificity shown in SEQ ID NO.7, and sequence is shown in SEQID NO.32.
10. as the detection method of each described deadly infectious disease virus in the claim 7～9, it is characterized in that the hybridization of detection probes described in the step 3 and described amplified production is carried out on solid phase carrier;
Described solid phase carrier comprises matrix and the adhesion layer that is fixed on the described matrix, and described detection probes is fixed on the described adhesion layer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102379771A CN102277452A (en) | 2011-08-18 | 2011-08-18 | Kit used for detecting highly infectious disease and detection method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102379771A CN102277452A (en) | 2011-08-18 | 2011-08-18 | Kit used for detecting highly infectious disease and detection method |
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| CN102277452A true CN102277452A (en) | 2011-12-14 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2011102379771A Pending CN102277452A (en) | 2011-08-18 | 2011-08-18 | Kit used for detecting highly infectious disease and detection method |
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Cited By (4)
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| CN108220481A (en) * | 2018-02-01 | 2018-06-29 | 淮安市疾病预防控制中心 | For detecting congo fever virus, three kinds of viral nucleic acid of library wave fever virus 1 and 2 types, kit and method simultaneously |
| CN111826463A (en) * | 2019-12-31 | 2020-10-27 | 深圳市人民医院 | Primer probe combination and kit for detecting five important arthropod/rodent-borne viruses and application of primer probe combination and kit |
| CN113308576A (en) * | 2021-06-10 | 2021-08-27 | 广州海关技术中心 | Kit for detecting rift valley fever virus based on digital PCR and detection method thereof |
| CN117737305A (en) * | 2023-09-28 | 2024-03-22 | 广州达安基因股份有限公司 | Group of molecular targets for detecting hemorrhagic fever pathogens and application thereof |
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| CN108220481A (en) * | 2018-02-01 | 2018-06-29 | 淮安市疾病预防控制中心 | For detecting congo fever virus, three kinds of viral nucleic acid of library wave fever virus 1 and 2 types, kit and method simultaneously |
| CN108220481B (en) * | 2018-02-01 | 2021-05-04 | 淮安市疾病预防控制中心 | Nucleic acid, kit and method for simultaneously detecting congo fever virus, kubo fever virus 1 and 2 types |
| CN111826463A (en) * | 2019-12-31 | 2020-10-27 | 深圳市人民医院 | Primer probe combination and kit for detecting five important arthropod/rodent-borne viruses and application of primer probe combination and kit |
| CN113308576A (en) * | 2021-06-10 | 2021-08-27 | 广州海关技术中心 | Kit for detecting rift valley fever virus based on digital PCR and detection method thereof |
| CN117737305A (en) * | 2023-09-28 | 2024-03-22 | 广州达安基因股份有限公司 | Group of molecular targets for detecting hemorrhagic fever pathogens and application thereof |
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