CN102286641A - Bioterrorism factor detection chip, detection kit and detection method - Google Patents

Abstract

The invention relates to a bioterrorism factor detection chip, detection kit and detection method. Probe molecules and PCR (polymerase chain reaction) amplification primers are designed according to gene conserved regions of multiple bioterrorism factors, and the probe molecules are integrated onto a detection chip, so that the detection flux is high; meanwhile, since the gene chip is used for detection, the invention has the advantages of high detection sensitivity and high speed; and according to analysis on designed PCR amplification primer sequences, a multiplex PCR technique can be used for amplifying nucleic acid substances of multiple bioterrorism factors within one time, thereby further enhancing the detection speed.

Description

The detection chip of the bio-terrorism factor, detection kit and detection method

[technical field]

The present invention relates to the biomolecule detection field, relate in particular to a kind of detection chip, detection kit and detection method of the bio-terrorism factor.

[background technology]

Traditional detection to the important bio-terrorism factor (as anthrax bacillus, lassa virus, clostridium botulinum toxin, brucella etc.) also rests on conventional biochemical cultivation stage, and this detection means can seem at a loss what to do in the face of the large-area bio-terrorism factor examination time; Same real-time fluorescence PCR technology can only increase simultaneously at a kind of or maximum several bio-terrorism factors usually, and in the face of containing a plurality of in sample even during tens bio-terrorism factors to be measured, just must carry out repeatedly PCR reaction could increase to the bio-terrorism factors all in the sample and reach testing goal, this just makes experimental implementation not only loaded down with trivial details time-consuming, the experiment consumptive material is caused greatly waste, and can not make examination and make judgement in time the bio-terrorism factor apace, easily lose the bio-terrorism factor in the sample is made the best moment that accurate result is analyzed.

[summary of the invention]

Based on this, be necessary to provide a kind of detection chip that is used for a plurality of bio-terrorism factors of rapid detection;

Simultaneously, also be necessary to provide a kind of detection kit that is used for a plurality of bio-terrorism factors of rapid detection;

Once more, also be necessary to provide a kind of detection method that is used for a plurality of bio-terrorism factors of rapid detection.

A kind of detection chip of the bio-terrorism factor is fixed with on it at least according to a kind of gene conservative region designed probe molecule in the multiple bio-terrorism factor; Described multiple bio-terrorism factor pair answers the designed probe molecular sequences as follows: Shigella: SEQ ID NO:1; Escherichia coli O 157: SEQ ID NO:2 or SEQ ID NO:3; Vibrio cholerae: SEQ IDNO:4; Ye Ersenshi bacillus: SEQ ID NO:5; Clostridium: SEQ ID NO:6; Salmonella: SEQ ID NO:7; Glanders Bai Kehuoerdeshi bacillus: SEQ ID NO:8; Pseudoglanders Bai Kehuoerdeshi bacillus: SEQ IDNO:9; Ornithosis virus: SEQ ID NO:10; Bai Neite Kao Keshi body: SEQ ID NO:11; Soil draws hot Francisella: SEQ IDNO:12; Rickettsia prowazeki: SEQ IDNO:13; Salmonella typhi: SEQ IDNO:14; Anthrax bacillus: SEQ ID NO:15; Brucella: SEQ ID NO:16; Ebola virus: SEQ ID NO:17; Lassa virus: SEQ ID NO:18; Marburg virus: SEQ ID NO:19; Nipah virus: SEQ ID NO:20; Hantaan virus: SEQ ID NO:21; Variola virus: SEQ ID NO:22; Machupo arenavirus: SEQ ID NO:23; Venezuelan equine encephalitis virus: SEQ ID NO:24; Eastern equine encephalitis virus: SEQ ID NO:25; Western equine encephalitis virus: SEQ ID NO:26.

Preferably, also be fixed with the positive quality control probe that nucleotides sequence is classified SEQ ID NO:81 as on the described detection chip.

A kind of detection kit of the bio-terrorism factor, comprise nucleic acid extraction liquid, hybridization reaction solution, PCR reaction solution, described PCR reaction solution comprise following many a pair of to primer centering at least: SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76, SEQ ID NO:77 and SEQ ID NO:78, SEQ ID NO:79 and SEQ ID NO:80.

Preferably, comprise that also the positive quality control primer is right in the described PCR reaction solution, the right forward primer of described positive quality control primer is SEQ ID NO:82, and reverse primer is SEQ ID NO:83.

A kind of detection method of the bio-terrorism factor comprises the steps: A, extracts the nucleic acid of testing sample; The nucleic acid of B, multiplex PCR amplification testing sample obtains pcr amplification product; C, with the assorted intersecting and merging color developing detection of the probe molecule on multiplex PCR amplified production and the detection chip.

Preferably, described testing sample is a Shigella, Escherichia coli O 157, vibrio cholerae, the Ye Ersenshi bacillus, Clostridium, salmonella, glanders Bai Kehuoerdeshi bacillus, pseudoglanders Bai Kehuoerdeshi bacillus, ornithosis virus, Bai Neite Kao Keshi body, soil draws hot Francisella, Rickettsia prowazeki, salmonella typhi, anthrax bacillus, brucella, Ebola virus, lassa virus, Marburg virus, Nipah virus, Hantaan virus, variola virus, machupo arenavirus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, the combination of one or more in the western equine encephalitis virus.

Preferably, described steps A specifically is bacterium, Rickettsiae, chlamydozoan and virus to be divided into two groups carry out nucleic acid extraction, and one group is bacterium, Rickettsiae and chlamydozoan, and another group is virus.

Preferably, described step B multiplex PCR specifically is the multiple bio-terrorism factor to be classified carry out pcr amplification respectively;

Described classification specifically is that the multiple bio-terrorism factor is divided into four groups, and first group comprises Shigella, Escherichia coli O 157, vibrio cholerae, Ye Ersenshi bacillus, Clostridium, salmonella; Second group comprises that glanders Bai Kehuoerdeshi bacillus, pseudoglanders Bai Kehuoerdeshi bacillus, ornithosis virus, Bai Neite Kao Keshi body, soil draw hot Francisella, Rickettsia prowazeki, salmonella typhi, anthrax bacillus, brucella; The 3rd group comprises Ebola virus, lassa virus, Marburg virus, Nipah virus, Hantaan virus; The 4th group comprises variola virus, machupo arenavirus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus.

According to the gene conserved regions design probe molecule and the pcr amplification primer of the multiple bio-terrorism factor, and a plurality of probe molecules are integrated on the detection chip, detect the flux height, simultaneously because the applying gene chip detection, detection sensitivity height, speed are fast; According to the analysis of pcr amplification primer sequence of design, adopt multiple PCR technique, can be once the nucleic acid substances of the multiple bio-terrorism factor be increased, thereby further accelerated the speed that detects.

Simultaneously, by design positive quality control control group the PCR process is carried out interpretation, avoid owing to the interference of PCR process mistake to detected result, experimental result more accurately and reliably.

[description of drawings]

Fig. 1 is a bio-terrorism factor detection method schema.

The detected result figure that Fig. 2 detects for the bio-terrorism factor.

[embodiment]

Below detection chip, detection kit and the detection method of the rapid detection bio-terrorism factor mainly are described in conjunction with specific embodiments.

By the analysis to common 25 kinds of bio-terrorism factor gene sequences, selecting conservative relatively zone is the target amplification region, and according to this regional sequence designing probe molecule and pcr amplification primer.At least be fixed with a kind of probe molecule on the detection chip, amplified production can be hybridized with fixed probe molecule on the detection chip, presents color by color reaction in the corresponding position of detection chip again.

Following table 1 is the probe molecule at 25 kinds of common bio-terrorism factor gene conserved regions design:

Table 1:

In preferred embodiment, detection chip is 96 orifice plate chips (96 orifice plate chips and preparation method thereof descriptions that see below, and separate case has been applied for a patent), is fixed with fixation layer at the bottom of the hole of 96 orifice plates, is fixed with probe molecule on the fixation layer; Preferably, fixation layer is a poly-l-lysine, and probe molecule is with 5*5, and latticed forms such as 6*6 are fixed on the fixation layer, and 96 orifice plates are polystyrene, polypropylene, acrylonitrile-styrene-butadienecopolymer or polyvinyl chloride material.

The detection chip that the specific probe molecule is arranged based on said fixing, comprise laboratory nucleic acid extraction liquid commonly used, hybridization reaction solution, hybridization washings etc. with the matching used test kit of detection chip, in addition, also comprise the PCR reaction solution, cover stoste and cover stoste diluent, colour generation stoste and colour generation stoste diluent etc.Test kit adopts multiple PCR technique, can increase at a plurality of bio-terrorism factors in the testing sample, need not to carry out repeatedly PCR and can finish all PCR reactions, the probe molecule that has designed on pcr amplification product and the detection chip is hybridized, carry out color reaction by reagent corresponding again, last application chip diagnostic platform scans the result.

Described hybridization reaction solution is to comprise 9.89% dextran sulfate sodium in the 1000mL distilled water solution, 0.199% maleic acid, 0.21%NaOH, 0.087%NaCl, 0.65% boric acid, 1%N, the two silica-based ethanamides of front three of O-.

The concrete prescription of described hybridization washings is: 0.6g NaCl, 0.7g NaH ₂PO ₄2H2O, 8.9g SDS (Sodium Dodecyl Sulfonate, sodium laurylsulfonate), 100mL 20 * SSC (saline sodium citrate, Trisodium Citrate) is dissolved in the 1000mL distilled water.Described 20 * SSC compound method is: 175.3g NaCl, and 88.2g Na3 Citrate is dissolved in 1000mLDEPC (diethylpyrocarbonate) water, and HCl regulates pH to 7.0, autoclaving.

It is that RNA ThermoScript II, the concentration of 10U/ μ L is the MgCl of 25mM that the PCR reaction solution removes the 4dNTPS comprise 10mM commonly used, archaeal dna polymerase, concentration that concentration is 5U/ μ L ₂At least also include the PCR primer that designs according to a kind of gene conservative region in 25 kinds of bio-terrorism factors outward Deng composition, every kind of a pair of upstream (Forward of all corresponding design of the bio-terrorism factor, F) and downstream (Reverse, R) primer, as shown in table 2 is the PCR primer sequence that 25 kinds of bio-terrorism factor pairs should design.

Table 2:

Preferably, also be fixed with the positive quality control probe on the detection chip, also comprise the positive quality control primer in the PCR reaction solution.Described positive quality control probe is SEQ ID NO:81, and positive quality control primer upstream primer is SEQ ID NO:82, and downstream primer is SEQID NO:83.When the pcr amplification condition setting is correct, nucleic acid-templated meeting is increased in a large number, corresponding probe is hybridized on pcr amplification product and the detection chip then, promptly can present color by corresponding position on a series of reaction chip again, can judge that the PCR process is accurately and reliably this moment, if color do not occur, represent that then there is mistake in the PCR process, need carry out the PCR reaction again.

Covering stoste is Streptavidin (the alkaline phosphatase-conjugated streptavidin that is marked with alkaline phosphatase, Strep-Ap) 1000 times of concentrated solutions, its function class is like secondary antibodies, and the Streptavidin that is marked with alkaline phosphatase can combine with the vitamin H on being marked at primer.Cover the stoste diluent, can cover on the detection chip, also have the prescription of blocking-up non-specific binding simultaneously not in conjunction with the blank parts of pcr amplification product; Covering the concrete prescription of stoste diluent is: 3.11g NaOH, and 150mL 0.1%Tween-20,10g BSA (N, the two silica-based ethanamides of front three of O-), 15g covers stoste, is dissolved in the 1000mL distilled water.Before the use, cover stoste and cover the mixed of stoste diluent with 1: 1000.

The composition of colour generation stoste is NBT/BCIP (NBT:Tetranitroblue 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 dark blue precipitate material simultaneously.Colour generation stoste diluent can guarantee that alkaline phosphatase activity is normal, and filling a prescription is: 2.4gNBT, 0.5gBCIP is dissolved in 100mLDMSO (dimethyl thioether) water.The prescription of colour generation stoste diluent is: 1.23g MgCl ₂6H ₂O, 1.32g NaCl, 0.8g HCl is dissolved in the 1000mL DMSO water; Before the use, colour generation stoste and colour generation stoste diluent were with 1: 50 mixed, and mixed solution is used to carry out color reaction.

Use the mentioned reagent box and can detect the terrified factor of 25 kinds of important biomolecules by color reaction, just which bio-terrorism factor in the terrified factor of described 25 kinds of detection of biological can be determined to contain in the testing sample according to probe points change in color on the detection chip, also can roughly judgement can be carried out to the content of examining the bio-terrorism factor in the testing sample simultaneously according to the depth of probe points colour-change.

Be illustrated in figure 1 as the detection method schema of the bio-terrorism factor, specifically comprise the steps:

S110: the nucleic acid that extracts testing sample: utilize the nucleic acid extraction liquid in the mentioned reagent box that the nucleic acid in the sample is extracted, the nucleic acid of carrying is used for further pcr amplification.

S120: the nucleic acid of multiplex PCR amplification testing sample: get step S110 gained nucleic acid extraction liquid, mix the PCR reaction solution and be mixed with a PCR system through pcr amplification, pcr amplification product is used for follow-up hybridization.

S130: with the assorted intersecting and merging color developing detection of the probe molecule on multiplex PCR amplified production and the detection chip: get above-mentioned steps S120 gained amplified production, hybridization reaction solution adds in the detection chip reacting hole, constant temperature hybridization; Add then and cover mixed solution (covering stoste and 1: 1000 the mixed solution that covers the stoste diluent) and colour generation mixed solution (1: 50 mixed solution of colour generation stoste and colour generation stoste diluent), can obtain each probe points colour-change situation on the detection chip through cleaning, color reaction.

Above-mentioned reacted chip can be obtained a result by visual inspection, also can judge, analyze the result simultaneously, obtains more detail analysis result.

The testing process and the result of the multiple bio-terrorism factor are described below by specific embodiment.

1, sample is prepared:

From the thing java standard library of having set up to be detected, find out samples such as the reference culture of above-mentioned 25 kinds of bio-terrorism factors and virus, rickettsia, chlamydozoan, because viral sample is difficult for obtaining and possessing hazardness, so adopt the method for synthetic, synthesize the specific fragment of viral nucleic acid and also clone as carrier with plasmid.

2, sample nucleic acid extraction:

Because bacterium, virus, rickettsia and chlamydial method for extracting nucleic acid are not quite similar, divide two pipes to carry out the extraction of sample nucleic acid at 1 testing sample, a bobbin is to bacterium, rickettsia and chlamydozoan, and another bobbin is to virus vector.Specific as follows:

1) get two 1.5mL centrifuge tubes, one is denoted as SI, and another is denoted as SII.Add the 0.2-1mL sample in the SI centrifuge tube, add the 0.2mL sample in the SII centrifuge tube in addition, two manage samples all with 8, centrifugal 5 minutes of 000g.Wherein the SI sample is according to following steps 2) carry out nucleic acid extraction to step 5), the SII sample is according to following steps 6) carry out nucleic acid extraction.

2) remove upper strata liquid, add 1mL 1 * E1 buffer in pipe, after in the concussion mode target throw out being suspended fully again, with 8, centrifugal 5 minutes of 000g.

3) remove upper strata liquid, add 50 μ L E2 buffer, after concussion mixed, heating was 10 minutes in boiling water.

4) move to ice bath after 2 minutes, after mixing with concussion again, with 8, centrifugal 5 minutes of 000g.

5) get 30 μ L upper stratas liquid (being the DNA of sample) in new aseptic Eppendorf tube, also label is SI, uses for follow-up PCR reaction.

6) nucleic acid of SII centrifuge tube sample extraction must be carried out the nucleic acid extraction with reference to High Pure Viral Nucleic Acid Purification Kit working instructions.

3, pcr amplification:

Above-mentioned sample nucleic acid extraction liquid need carry out pcr amplification can be used for hybridization, owing to have competition and interference between the PCR primer of described 25 kinds of bio-terrorism factors, so PCR reaction solution in the described test kit is divided into Mix1, Mix2, four parts of Mix3, Mix4.Shown in the pairing bio-terrorism factor of PCR primer among the Mix1 234 and other compositions table 3 specific as follows, 4,5,6:

Table 3:Mix1:

Table 4:Mix2:

Table 5:Mix3

Table 6:Mix4:

The concrete steps of pcr amplification are as follows:

1) according to the institute of above-mentioned PCR primer at the bio-terrorism factor, SI pipe sample nucleic acid extraction liquid need be divided into two pipe PCR and react.The PCR that gets 200 μ L manages two, is denoted as Tube 1 and Tube 2 respectively.

2) in Tube 1, add 41.5 μ L Ml, 0.5 μ L archaeal dna polymerase, 8 μ L SI pipe sample nucleic acid extraction liquid; Add 41.5 μ L M2,0.5 μ L archaeal dna polymerase, 8 μ L SI pipe sample nucleic acid extraction liquid among the Tube2, after interpolation finishes Tube 1 and 2 liang of pipes of Tube are inserted in the PCR reaction instrument.

3) according to following program setting PCR reaction conditions: first 95 ℃ of 5min, 95 ℃ of 30sec then, 56 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations, last 72 ℃ of 5min, the reaction products therefrom is used for follow-up hybridization.

4) with above-mentioned SI tubing seemingly, SII pipe sample nucleic acid extraction liquid also need be divided into two pipe PCR reactions.The PCR that gets 200 μ L manages two, is denoted as Tube 3 and Tube 4 respectively.

5) in Tube 3, add 41.5 μ L M3,0.5 μ L archaeal dna polymerase, 0.25 μ L AMV ThermoScript II, 8 μ L SII pipe sample nucleic acid extraction liquid; Add 41.5 μ L M4,0.5 μ L archaeal dna polymerase, 0.25 μ L AMV ThermoScript II, 8 μ L SII pipe sample nucleic acid extraction liquid among the Tube 4, after interpolation finishes Tube 3 and 4 liang of pipes of Tube are inserted in the PCR reaction instrument.

6) according to following program setting PCR reaction conditions: first 42 ℃ of 40min, 95 ℃ of 5min, 95 ℃ of 30sec then, 56 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations, 72 ℃ of 5min, the reaction products therefrom is used for follow-up hybridization.

4, hybridization:

Before carrying out hybridization, guarantee pcr amplification product and hybridize washings to rise again to room temperature.Hybridization carries out in thermostat container, before the reaction thermostat container is preheated to 45 ℃.The hybridization concrete steps are as follows:

1) with the pcr amplification product among the embodiment 3, promptly Tube 1～4 grade four is managed, and after being heated to 95 ℃ of sex change in 5 minutes on the PCR instrument, taking-up places on ice.

2) the pcr amplification product 20 μ L that respectively get sex change from four pipes of above-mentioned steps join in the reacting hole of described detection chip, add hybridization reaction solution 200 μ L again, stick transparent adhesive film, put into 45 ℃ of thermostat container hybridization 1.5h.

5, colour generation is covered in cleaning:

After above-mentioned steps 4 hybridizations finish, take out detection chip, the film that removes photoresist, liquid in the evacuation apertures, clean according to following steps and to cover colour generation:

1) in the reacting hole of detection chip, adds 200 μ L hybridization washings, firmly rocked detection chip 20 seconds (do not overflow with liquid in the reacting hole and exceed), firmly dry liquid in the reacting hole then, repeat above-mentioned action 2 times.

2) in reacting hole, add 200 μ L and cover mixed solution (cover stoste with cover damping fluid mixed) with 1: 1000, leave standstill 10min after, firmly dry liquid in the reacting hole.

3) in reacting hole, add 200 μ L colour generation mixed solutions (colour generation stoste is mixed with 1: 50 with the colour generation damping fluid), firmly rock 20 seconds (do not overflow exceed with liquid in the reacting hole), firmly dry liquid in the reacting hole then, repeat above-mentioned action 2 times.

4) Yu Kongzhong adds 200 μ L colour generation mixed solutions (colour generation stoste is mixed with 1: 50 with the colour generation damping fluid), leave standstill 10min after, firmly dry liquid in the hole.

6, interpretation as a result:

After above-mentioned steps 5 reactions finished, each probe points change in color in the hole that detects by an unaided eye as shown in Figure 2, was analyzed the contained bio-terrorism factor in the sample according to situation about changing.

According to the gene conserved regions design probe molecule and the pcr amplification primer of the multiple bio-terrorism factor, and a plurality of probe molecules are integrated on the detection chip, detect the flux height, simultaneously because the applying gene chip detection, detection sensitivity height, speed are fast; According to the analysis of pcr amplification primer sequence of design, adopt multiple PCR technique, can be once the nucleic acid substances of the multiple bio-terrorism factor be increased, thereby further accelerated the speed that detects.

Simultaneously, by design positive quality control control group the PCR process is carried out interpretation, avoid owing to the interference of PCR process mistake to detected result, experimental result more accurately and reliably.

The above embodiment has only expressed one or more embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

SEQUENCE?LISTING

＜110〉Shenzhen Bo Ermei bio tech ltd

The detection chip of＜120〉the bio-terrorism factor, detection kit and detection method

<160>83

<170>PatentIn?version?3.3

<210>1

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>1

acgtgaagtg?gtcagaagc 19

<210>2

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>2

acaacacttg?atgatctcag?gg 23

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>3

atacaatgac?cagagatgca?t 21

<210>4

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>4

agagcttaat?gcggagacgg?cccaaaaat 29

<210>5

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>5

ccatctttcc?gcatcaacga?atatg 25

<210>6

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>6

cttgcatagc?ctagagatag?gtgaag 26

<210>7

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>7

ttctacattg?acagaatcct?c 21

<210>8

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>8

catacggatg?tatagaacca?at 22

<210>9

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>9

cgccgcaaga?cgccatcgtt?cat 23

<210>10

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>10

tcattgtcat?tatggtgatt?cagga 25

<210>11

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>11

tgtatggttc?caatgttgtg?ctg 23

<210>12

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>12

agatactgct?gctgctcaga?cag 23

<210>13

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to use probe as hybridizing

<400>13

actttactta?tgatccgggt?tttatg 26

<210>14

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to use probe as hybridizing

<400>14

tggagacttc?ggtcgcgtag 20

<210>15

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<220>

<221>misc_feature

<222>(10)..(10)

<223>n?is?a，c，g，or?t

<400>15

gtagagggtn?actctgccgg?t 21

<210>16

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>16

tcagacgttg?cctattgggc?c 21

<210>17

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>17

aggatggagc?tttcttcctc?tatga 25

<210>18

<211>33

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>18

aatgcagagt?tgctcaataa?tcagttcggg?acc 33

<210>19

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>19

cacacacagt?cagacactag?ccgtcct 27

<210>20

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>20

gaacagagtg?gcagacagtc 20

<210>21

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>21

ataacccgcc?atgaacaaca?gcttg 25

<210>22

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>22

acacgtctgt?tggagacgt 19

<210>23

<211>30

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>23

ggctgatata?gtgccaattc?cacagggtct 30

<210>24

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>24

agatacccta?tgtccaccat?cctgg 25

<210>25

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>25

ggtagtctat?tactacaaca?gatac 25

<210>26

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>26

gtggggcgag?aagggctgga?gtacg 25

<210>27

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>27

tagaaggcag?agatggaaga?gtt 23

<210>28

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>28

gccggtcagc?caccctctga?gagtac 26

<210>29

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>29

ggacagcagt?tataccactc?tgc 23

<210>30

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>30

ctgatgatgg?caattcagta?t 21

<210>31

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>31

gccacagcaa?caacgcaac 19

<210>32

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to specific nucleotide sequence design, with primer as pcr amplification

<400>32

agagaacgaa?cttcatccgc?tac 23

<210>33

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>33

ggtcatggtg?atgttgatta?ctat 24

<210>34

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>34

tcgataccct?gcaccaagca 20

<210>35

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>35

aaaactcaaa?tgaattgacg?g 21

<210>36

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>36

gtacaaggcc?cgggaacgta?ttcaccgtg 29

<210>37

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>37

gctcgtttac?gacctgaatt?ac 22

<210>38

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>38

agacggctgg?tactgatcga?taatgcca 28

<210>39

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>39

cagttgattc?tcccacc 17

<210>40

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>40

tgtcttgttg?agcatgaga 19

<210>41

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>41

atcgaatcag?ggcgttcaag 20

<210>42

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>42

cattcggtga?cgacacgacc 20

<210>43

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>43

gccatcatgc?tgtttcgtt?t 21

<210>44

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>44

cggcgtgcca?cttgaga 17

<210>45

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to specific nucleotide sequence design, with primer as pcr amplification

<400>45

actcaacgca?ctggaaccgc 20

<210>46

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>46

tagctgaagc?caattcgcc 19

<210>47

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to specific nucleotide sequence design, with primer as pcr amplification

<400>47

gcaggtttag?cragctgttc?tac 23

<210>48

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>48

ggagcytgcc?attgtaatcttac 23

<210>49

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>49

tcggtaaaga?tgtaatcgat?ataag 25

<210>50

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>50

catatcctcg?ataccataat?atgc 24

<210>51

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>51

actgctaaaa?ccactact 18

<210>52

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>52

ttaacgcagt?aaagagag 18

<210>53

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>53

cagaggcatt?tgaaaagttc 20

<210>54

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>54

acgttaataa?tagggtacg 19

<210>55

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>55

tggctcggtt?gccaatatca?a 21

<210>56

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>56

cgcgcttgcc?ttcaggtct?g 21

<210>57

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>57

aatgggctga?aaattgctac?aatc 24

<210>58

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>58

tttttttagt?ttcccagaag?gcccact 27

<210>59

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to specific nucleotide sequence design, with primer as pcr amplification

<400>59

ggggctcggg?ctgggagaga?tggag 25

<210>60

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>60

ctgcccctgt?tttgtcagac?atgcc 25

<210>61

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>61

caattccacc?ttcagaaaac?tg 22

<210>62

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>62

gctaattttt?ctcgtttctg?gct 23

<210>63

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>63

ggctagagag?gcaaaatttg?ctgc 24

<210>64

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>64

accggatgtg?ctcacagaac?t 21

<210>65

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>65

aaaaactggg?atgagtgact?tg 22

<210>66

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>66

ttctgcatcc?tgagctttt?gtc 23

<210>67

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>67

cggaagagac?ggtgtragaa?tatgt 25

<210>68

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>68

cgtgtaacac?gactcacaat?agaatct 27

<210>69

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>69

ccatttttga?agcccttctc?atcatg 26

<210>70

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>70

caaattcttg?ggagatcttg?ggacaacac 29

<210>71

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>71

gttttgggca?caggaaacag?c 21

<210>72

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>72

ttggctcggc?atcgtgttcg?cg 22

<210>73

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>73

accacctggg?agtccttgga 20

<210>74

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>74

tggctggtga?atccattcct 20

<210>75

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>75

accacctggg?agtccttgga 20

<210>76

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>76

ttggctcggc?atcgtgttcg?cf 22

<210>77

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>77

cacatggatg?gccgcacgaa 20

<210>78

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>78

cagcaaagta?acgccaggagg?agta 25

<210>79

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>79

cgagcagacg?caacagcaga?a 21

<210>80

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>80

caggatagca?agagcgacac?ca 22

<210>81

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, to be used as hybridization probe

<400>81

atcgttcgtg?acatcaagga?gaa 23

<210>82

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>82

aaaactcaaa?tgaattgacg?g 21

<210>83

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification

<400>83

gtacaaggcc?cgggaacgta?ttcaccgtg 29

Claims (8)

1. the detection chip of a bio-terrorism factor is characterized in that, is fixed with at least on the described detection chip according to a kind of gene conservative region designed probe molecule in the multiple bio-terrorism factor; Described multiple bio-terrorism factor pair answers the designed probe molecular sequences as follows:

Shigella: SEQ ID NO:1; Escherichia coli O 157: SEQ ID NO:2 or SEQ ID NO:3; Vibrio cholerae: SEQ ID NO:4; Ye Ersenshi bacillus: SEQ ID NO:5; Clostridium: SEQ ID NO:6; Salmonella: SEQ ID NO:7; Glanders Bai Kehuoerdeshi bacillus: SEQ ID NO:8; Pseudoglanders Bai Kehuoerdeshi bacillus: SEQ ID NO:9; Ornithosis virus: SEQ ID NO:1Q; Bai Neite Kao Keshi body: SEQ ID NO:11; Soil draws hot Francisella: SEQ ID NO:12; Rickettsia prowazeki: SEQ ID NO:13; Salmonella typhi: SEQID NO:14; Anthrax bacillus: SEQ ID NO:15; Brucella: SEQ ID NO:16; Ebola virus: SEQID NO:17; Lassa virus: SEQ ID NO:18; Marburg virus: SEQ ID NO:19; Nipah virus: SEQ IDNO:2Q; Hantaan virus: SEQ ID NO:21; Variola virus: SEQ ID NO:22; Machupo arenavirus: SEQ ID NO:23; Venezuelan equine encephalitis virus: SEQ ID NO:24; Eastern equine encephalitis virus: SEQ ID NO:25; Western equine encephalitis virus: SEQ ID NO:26.

2. the detection chip of the bio-terrorism factor as claimed in claim 1 is characterized in that, also is fixed with the positive quality control probe that nucleotides sequence is classified SEQ ID NO:81 as on the described detection chip.

3. the detection kit of a bio-terrorism factor, comprise nucleic acid extraction liquid, hybridization reaction solution, the PCR reaction solution, it is characterized in that described PCR reaction solution comprises following many a pair of to primer centering at least: SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76, SEQ ID NO:77 and SEQ ID NO:78, SEQ ID NO:79 and SEQ ID NO:8Q.

4. the detection kit of the bio-terrorism factor as claimed in claim 3 is characterized in that, comprises that also the positive quality control primer is right in the described PCR reaction solution, and the right forward primer of described positive quality control primer is SEQ ID NO:82, and reverse primer is SEQID NO:83.

5. the detection method of a bio-terrorism factor is characterized in that, comprises the steps:

The nucleic acid of A, extraction testing sample;

The nucleic acid of B, multiplex PCR amplification testing sample obtains pcr amplification product;

C, with the assorted intersecting and merging color developing detection of the probe molecule on multiplex PCR amplified production and the detection chip.

6. the detection method of the bio-terrorism factor as claimed in claim 5, it is characterized in that described testing sample is a Shigella, Escherichia coli O 157, vibrio cholerae, the Ye Ersenshi bacillus, Clostridium, salmonella, glanders Bai Kehuoerdeshi bacillus, pseudoglanders Bai Kehuoerdeshi bacillus, ornithosis virus, Bai Neite Kao Keshi body, soil draws hot Francisella, Rickettsia prowazeki, salmonella typhi, anthrax bacillus, brucella, Ebola virus, lassa virus, Marburg virus, Nipah virus, Hantaan virus, variola virus, machupo arenavirus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, the combination of one or more in the western equine encephalitis virus.

7. the detection method of the bio-terrorism factor as claimed in claim 5, it is characterized in that, described steps A specifically is bacterium, Rickettsiae, chlamydozoan and virus to be divided into two groups carry out nucleic acid extraction, and one group is bacterium, Rickettsiae and chlamydozoan, and another group is virus.

8. the detection method of the bio-terrorism factor as claimed in claim 5 is characterized in that, described step B multiplex PCR specifically is the multiple bio-terrorism factor to be classified carry out pcr amplification respectively;

Described classification specifically is that the multiple bio-terrorism factor is divided into four groups, and first group comprises Shigella, Escherichia coli O 157, vibrio cholerae, Ye Ersenshi bacillus, Clostridium, salmonella; Second group comprises that glanders Bai Kehuoerdeshi bacillus, pseudoglanders Bai Kehuoerdeshi bacillus, ornithosis virus, Bai Neite Kao Keshi body, soil draw hot Francisella, Rickettsia prowazeki, salmonella typhi, anthrax bacillus, brucella; The 3rd group comprises Ebola virus, lassa virus, Marburg virus, Nipah virus, Hantaan virus; The 4th group comprises variola virus, machupo arenavirus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus.

Images