CN105087824A - Preparation and application of hemorrhagic fever associated pathogen identifying gene chip - Google Patents

Preparation and application of hemorrhagic fever associated pathogen identifying gene chip Download PDF

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CN105087824A
CN105087824A CN201510096044.3A CN201510096044A CN105087824A CN 105087824 A CN105087824 A CN 105087824A CN 201510096044 A CN201510096044 A CN 201510096044A CN 105087824 A CN105087824 A CN 105087824A
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virus
probe
hemorrhagic fever
quality control
gene chip
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CN105087824B (en
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张严峻
颜浩
王升启
刘琪琦
陈苏红
刘志红
孙晓彦
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Institute of Radiation Medicine of CAMMS
Zhejiang Center for Disease Control and Prevention
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Institute of Radiation Medicine of CAMMS
Zhejiang Center for Disease Control and Prevention
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to a hemorrhagic fever associated pathogen identifying gene chip; the preparation method comprises preparation of a specific primer, preparation of a pathogen specific oligonucleotide probe, preparation of an oligonucleotide chip, establishment of an RT-PCR (reverse transcription-polymerase chain reaction) system and establishment of a hybrid system and a signal detection method. The gene chip prepared by the invention can be used for simultaneously identifying 16 hemorrhagic fever associated pathogen microorganisms, including Zaire Ebola virus, Sudan Ebola virus, marburg virus, lassa virus, junin virus, Machupo virus, rift valley fever virus, Crimea-Congo hemorrhagic fever virus, plasmodium, hantaan virus, SFTS (severe fever with thrombocytopenia syndrome) virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A virus and influenza B virus. The gene chip has the characteristics of being rapid and accurate, high in throughput and high in sensitivity; and a new technological means is offered for the diagnosis of hemorrhagic fever pathogen, health supervision and the control and prevention of infectious diseases.

Description

The preparation of hemorrhagic fever related diseases substance sldh gene chip and purposes
Technical field
The present invention relates to preparation and purposes that 16 kinds of hemorrhagic fever related diseases pathogen nucleic acid detect gene chip, belong to gene chip detecting technique field.
Background technology
Viral hemorrhagic fever (ViralHemorrhagicFever, VHF) is one group to be caused by dissimilar virus, to generate heat, hemorrhage one group of disease of natural focus for main clinic symptoms.Hemorrhagic fever virus is mainly distributed in inovirus section (Filoviridae), Arenaviridae (Arenaviridae), bunyaviridae (Bunyaviridae) and flaviviridae (Flaviviridae).The viral species that can cause blood-head is various, wherein affects larger comprising in the world: inovirus section Ebola virus, Marburg virus, Arenaviridae lassa virus, the peaceful virus of turtledove, Machupo virus, bunyaviridae Rift Valley fever virus, crimean-Congo hemorrhagic fever virus, Hantaan virus, heating companion thrombocytopenic syndromes virus, flaviviridae dengue virus, yellow fever virus, Togaviridae Chikungunya virus etc.The clinical manifestation at such disease incidence initial stage is not special, similar influenza, malaria, often can develop into subcutaneous, body cavity and visceral hemorrhage, and the consequent is shock, stupor and neurological dysfunction etc., and case fatality rate is high, and harm is serious.
Hemorrhagic fever virus host and media types extensive, circulation way is varied, along with the progress of economic globalization, each department commerce and trade contact and personnel transfer are more quick frequent, the geographical boundary of host and medium distribution is broken, thus cause the expansion of epidemic situation Epidemic Scope and break out, too increase the possibility that Introduced cases epidemic situation occurs.The ebola hemorrhagic fever that West Africa in 2014 is broken out is the acute hemorrhage sexually transmitted disease caused by Ebola virus, from in March, 2014 report occur first batch of case to December 24 have 19497 people doubtful or confirm infect, cause 7588 people dead, be the maximum and the most complicated Ebola's epidemic situation occurred since Late Cambrian Ebola virus for 1976, the case that this epidemic situation occurs and death figure have exceeded the summation of other epidemic situations all.Ebola virus has been classified as one of the most serious virus of mankind's harm by WHO, its average case fatality rate is about 50%, the case fatality rate occurred in epidemic situation is not in the past from 25% to 90% etc., find that there is five hypotypes at present, wherein Zaire's type and the Sudan's type case fatality rate the highest, 88.8% and 53.2% can be reached respectively.Chinese Guangdong dengue outbreak in 2014, to the accumulative report in Guangdong on December 1 cases of dengue fever 45053 example.According to estimates, the case of the annual actual infection dengue virus in the whole world is up to 3.9 hundred million, wherein dead because of severe infections more than 20,000 people.The viral epidemic situation not only previously found is broken out frequently, causes the new virus of emerging infectious disease to be also constantly found.Within 2010, China finds the novel bunyavirus with important public health meaning---heating companion thrombocytopenic syndromes virus (SevereFeverwithThrombocytopeniaSyndromeVirus, SFTSV), the heating companion thrombocytopenic syndromes that this virus-infected human causes, be a kind of emerging infectious disease being main clinical manifestation with generate heat companion white corpuscle, thrombopenia and multi-organ function injury, mortality ratio is up to 30%.At present in Shandong, Hubei, Zhejiang, multiple provinces and cities such as Jiangsu find SFTSV cases of infection, 2011-2013, Missouri, USA, Japan, Korea S also report the similar SFTSV virus infection case finding that there is and propagate through tick sting in succession, and SFTSV infects becomes global important public hygiene problem gradually.
Because viral hemorrhagic fever case fatality rate is high, route of transmission is wide, and very easily cause society panic, hemorrhagic fever virus is classified as class biological weapon by disease prevention and control center of the U.S., and therefore the prevention and control of viral hemorrhagic fever and treatment means seem particularly important.But business-like hemorrhagic fever vaccine only has HFRS inactivated vaccine and Yellow Fever Vaccine at present, still has Argentinian hemorrhagic fever vaccine and Rift Valley fever vaccine to be in clinical experimental stage in addition; At drug treatment, there is no the specific medicament for viral hemorrhagic fever." examine ", " preventing ", " controlling " three links constitute complete prevention and control of diseases chain, wherein " examine " and be not only a wherein indispensable crucial ring, and be the prerequisite of effectively control.In the shortage situation of the occurred frequently of viral hemorrhagic fever with clinical treatment means, pathogenic agent is diagnosed to seem particularly important accurately and effectively.
Because most the infected lacks special clinical manifestation in early days, and all there is similar clinical symptom between different viral hemorrhagic fevers or between viral hemorrhagic fever to other diseases (such as malaria, influenza etc.), laboratory diagnostic method must be relied on and confirmed.Hemorrhagic disease substance test in laboratory method conventional at present mainly comprises virus purification, polymerase chain reaction method (PCR), enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IFA), electron microscopy (EM) and immunohistochemistry technique.Aforesaid method has respective relative merits, some method complicated operations, time and effort consuming; Some method specificitys and/or susceptibility lower; Some method sample consumptions are too much; And all there is detection flux deficiency in aforesaid method, the problem that sensing range is limited.
Gene chip (Genechip) is also called DNA chip (DNAchip), DNA microarray (DNAmicroarray), the one of biochip, be also develop in biochip technology the most ripe, at first enter application and realize commercial technology.Biochip technology refers to and directly goes up fabricated in situ oligonucleotide pair in support surface by DNA probe or at solid support (as slide, silicon chip, nylon membrane etc.), then with mark sample hybridization to be analyzed, the sample of mark passes through the DNA fragmentation Complementary hybridization of known base sequence on gene chip, thus obtain the genetic information of sample, determine the nucleotide sequence of sample, or gene expression amount and characteristic thereof are analyzed.Biochip technology because of its have fast, accurately, the feature such as high-throughput, high-level efficiency, low cost is widely used in the aspects such as expression pattern analysis, single nucleotide polymorphism analysis, medical diagnosis on disease, the pathogenic microorganism examination.
This research, in conjunction with the actual needs of blooding diagnosis, have chosen 13 kinds of hemorrhagic fever correlated virus, and adds the plasmodium and influenza virus that can cause similar symptoms, is intended to develop blood-head related diseases substance sldh gene chip.Based on chemical luminescence detection method, detect 16 kinds of hemorrhagic fever related diseases substances simultaneously, comprise Zaire type Ebola virus, the Sudan type Ebola virus, Marburg virus, lassa virus, the peaceful virus of turtledove, Machupo virus, Rift Valley fever virus, crimean-Congo hemorrhagic fever virus, plasmodium, Hantaan virus, heating companion thrombocytopenic syndromes virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A, Type B influenza virus.This chip method specificity is high, and susceptibility is strong, is suitable for the multiple Rapid identification causing febrile conditions pathogenic agent.
Summary of the invention
The object of the present invention is to provide a kind of gene chip, this chip can detect 16 kinds of hemorrhagic fever related diseases substances simultaneously, comprise Zaire type Ebola virus, the Sudan type Ebola virus, Marburg virus, lassa virus, turtledove is rather viral, Machupo virus, Rift Valley fever virus, crimean-Congo hemorrhagic fever virus, plasmodium, Hantaan virus, heating companion thrombocytopenic syndromes virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A, Type B influenza virus, have fast, accurately, high-throughput, highly sensitive feature, can be the diagnosis of hemorrhagic fever cause of disease, sanitary inspection and transmissible disease prevention and control provide a kind of new technique means.
Gene chip its preparation method of the present invention comprises the steps:
1. prepare Auele Specific Primer
Through each pathogen gene group of comparison, select special, conservative fragments as detection target gene, under the prerequisite ensureing the amplification of each pathogenic agent target gene specific, taking into account its sensitivity, therefore pathogen specific primer is carried out multiplely be in charge of combination, finally determining the multiple TR-PCR system of 3 pipe through optimizing.The amplified target cause of disease kind of preferred 29 Auele Specific Primers and correspondence thereof and to be in charge of combined situation as shown in table 1, each downstream primer 5 ' holds mark vitamin H.
The amplified target cause of disease kind of table 1 hemorrhagic fever related diseases substance primer and correspondence thereof and be in charge of combined situation
2. prepare specific probe
In line with conservative between type, belong between special principle, according to the comparison between 16 kinds of related diseases substance target-gene sequences, the relatively special district of the sequence within the scope of upstream and downstream primer carries out the design of specific probe.The corresponding specific oligonucleotide probe of often kind of target cause of disease, preferred specific probe sequence is as shown in table 2, and 3 ' end of every bar probe adds 12 base T and at 3 ' end with amido modified.
The target cause of disease of table 2 specific probe sequence and correspondence
3. prepare gene chip
A preferred embodiment, in step 2, each oligonucleotide probe is when point sample, is diluted to final concentration 50 μMs with chip sampling liquid (6 × SSC, 5% glycerine, 0.1%SDS).In contact point sample mode, probe points is modified on slide (array is as shown in table 3) to the aldehyde radicalization of blank with commercially available gene chip sample applying instrument, the point sample amount of each probe is 3nl, wherein Quality Control probe is 20T sequence, 5 ' end biotin labeling, 3 ' Amino End Group are modified, and are used for monitoring aldehyde radical blade matrix amount.Point process processed keep certain humidity, oligonucleotide chip preparation after, chip should at least be placed 24 hours in drying at room temperature, make probe and carrier full cross-linked.
Table 3 oligonucleotide probe array
Quality Control Quality Control Quality Control Quality Control Quality Control Quality Control Quality Control Quality Control
ZEBO-P SEBO-P MAR-P LF-P JUN-P MAC-P RVF-P CCHFV-P
ZEBO-P SEBO-P MAR-P LF-P JUN-P MAC-P RVF-P CCHFV-P
ZEBO-P SEBO-P MAR-P LF-P JUN-P MAC-P RVF-P CCHFV-P
MAL-P HV-MP SFTSV-P DEN-P YFV-P CHIKV-P FluA-P FluB-P
MAL-P HV-MP SFTSV-P DEN-P YFV-P CHIKV-P FluA-P FluB-P
MAL-P HV-MP SFTSV-P DEN-P YFV-P CHIKV-P FluA-P FluB-P
Blank Blank Blank Blank Blank Blank Blank Blank
4. set up RT-PCR system
In gene chip of the present invention, the feature of RT-PCR system is asymmetric multiple RT-PCR reaction system.Suitable RT-PCR system can improve the detection sensitivity of chip further.The factors such as the consumption of the absolute concentration of labeled primer and non-marked primer and relative proportion, enzyme are optimized.Preferred RT-PCR system and amplification condition, as shown in table 4:
The asymmetric multiple RT-PCR system formulation of table 4-1
The preferred RT-PCR amplification condition of table 4-2
5. set up chip hybridization system
Suitable hybridization system also has very large promoter action to the specificity of chip and sensitivity improving.By optimizing the pre-washing lotion and hybridization solution composition, hybridization conditions and the post-hybridization wash conditions that obtain and can ensure specificity and sensitivity simultaneously.Be placed in pre-washing lotion (0.2%SDS) before gene chip hybridization respectively and clean 20s with deionized water, RT-PCR product is placed in ice-water bath immediately after 95 DEG C of sex change 5min.Mixed with hybridization solution equal-volume by RT-PCR product, each component of preferred hybridization solution is 8 × SSC, 10% methane amide, 1.2%SDS, 10 × Denhardt ' s.Preferred hybridization conditions is that 45 DEG C of water-baths hybridize 1 hour.Preferred wash conditions is respectively in washing lotion A (1 × SSC, 0.2%SDS), respectively washs 20s in washing lotion B (0.2 × SSC) and washing lotion C (0.1 × SSC).
6. set up signal detecting method
Diluted with 1: 1000 by marking fluid (Streptavidin-HRP) diluent (1 × PBS+0.1%BSA), add 15 μ l in each hybridization region, chip is placed in 37 DEG C and hatches 30min.Clean 20s by PBST washing lotion (1 × PBS+0.05%Tween-20) after taking out, dry.By chemiluminescent HRP substrate A liquid and the mixing of B liquid (Millipore company) equal-volume, each hybridization region adds 20 μ l and expansion paving is even rapidly, is placed in chemiluminescence imaging instrument immediately and scans.Record result, judges result according to strength of signal.
The hemorrhagic fever related diseases substance sldh gene chip more than prepared comprises oligonucleotide chip, RT-PCR system, hybridization solution, washing lotion A, washing lotion B, washing lotion C, Streptavidin-HRP, diluent, PBST washing lotion, chemiluminescent HRP substrate A liquid and B liquid.
The present invention establishes a kind of gene chip based on chemiluminescence platform, can distinguish 16 kinds of hemorrhagic fever related diseases substances, comprise Zaire type Ebola virus, the Sudan type Ebola virus, Marburg virus, lassa virus, the peaceful virus of turtledove, Machupo virus, Rift Valley fever virus, crimean-Congo hemorrhagic fever virus, plasmodium, Hantaan virus, heating companion thrombocytopenic syndromes virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A, Type B influenza virus.This gene chip and corresponding preparation method, have stronger practicality, realizes multiple sick detection of planting, while shortening sense cycle, improve detection efficiency by an experimental implementation.Compare with conventional culture methods, there is quick, accurate, sensitive advantage, compare with other nucleic acid detection methods with immunological method, there is the advantage of high-throughput, high specific, can be the diagnosis of hemorrhagic fever cause of disease, sanitary inspection and transmissible disease prevention and control and a kind of new technique means is provided.
Accompanying drawing explanation
Fig. 1: gene chip schematic appearance, often opens and chip has 10 identical hybridization districts being distributed with probe dot matrix.
Fig. 2: hemorrhagic fever related diseases substance sldh gene chip probe dot matrix layout, wherein 1a to 8a is Quality Control point; 1b to 1d is Zaire's type Ebola virus probe; 2b to 2d is the Sudan's type Ebola virus probe; 3b to 3d is Marburg virus probe; 4b to 4d is lassa virus probe; 5b to 5d is the peaceful Viral Probe of turtledove; 6b to 6d is Machupo virus probe; 7b to 7d is Rift Valley fever virus probe; 8b to 8d is crimean-Congo hemorrhagic fever virus probe; 1e to 1g is plasmodium probe; 2e to 2g is Hantaan virus probe; 3e to 3g is heating companion thrombocytopenic syndromes Viral Probe; 4e to 4g is dengue virus probe; 5e to 5g is yellow fever virus probe; 6e to 6g is Chikungunya virus probe; 7e to 7g is influenza A probe; 8e to 8g is Type B influenza virus probe; 1h to 8h is blank.
Fig. 3: hemorrhagic fever related diseases substance sldh gene chip Evaluation on specificity result figure, A is Ebola virus Zaire type; B is Ebola virus the Sudan type; C is Marburg virus; D is lassa virus; E is that turtledove is rather viral; F is Machupo virus; G is Rift Valley fever virus; H is crimean-Congo hemorrhagic fever virus; I is plasmodium falciparum; J is every other day cruel protozoon; K is Hantaan virus; L is heating companion thrombocytopenic syndromes virus; M is dengue virus; N is yellow fever virus; O is Chikungunya virus; P is influenza A virus; Q is influenza virus B type; R is negative control.
Fig. 4: hemorrhagic fever related diseases substance sldh gene chip sensitivity chip detection result figure, for Ebola virus, plasmodium and heating companion thrombocytopenic syndromes virus.Wherein in figure, A1 ~ A5 represents Ebola virus Zaire type sensitivity evaluation result; B1 ~ B5 represents Ebola virus the Sudan type sensitivity evaluation result; C1 ~ C5 represents plasmodium sensitivity evaluation result; D1 ~ D5 representative heating companion thrombocytopenic syndromes viral sensitivity evaluation result.1 ~ 5 represents 10 successively 4copies/ μ l, 10 3copies/ μ l, 10 2copies/ μ l, 10 1copies/ μ l and negative control.
Embodiment
The following example is intended to illustrate instead of limit the present invention.
Embodiment 1: the development of hemorrhagic fever related diseases substance sldh gene chip
One, the design of Auele Specific Primer and screening
Log in NCBI website, the whole genome sequence retrieving correlated virus genus virus at GenBank compares, and plasmodium has been downloaded 4 kinds of plasmodial 18srRNA genes and compared.The AlignX program in VectorNTIAdvance10 (invitrogen) software package is used to carry out overall comparison according to the optimum configurations of acquiescence to each pathogen gene sequence.According to the conserved regions design Auele Specific Primer of comparison result in gene order, finally determine 29 Auele Specific Primers (as shown in table 1) through screening, downstream primer is carried out biotin labeling, as the primer that chip uses.
Two, the structure of reference material
Except part hemorrhagic fever related diseases substance obtains sample of nucleic acid, all the other pathogenic agent (being virus) all do not obtain suitable sample, for satisfied experiment demand, this research adopts the pathogen gene group sequence of the artificial constructed free nucleic acid sample of method of overlap-extension PCR.Carry out in-vitro transcription according to the step of ThermoTranscriptAidT7HighYieldTranscriptionKit specification sheets to the virus particle built, preparation in-vitro transcription RNA is also quantitative, as specificity and sensitivity reference material.
Three, the design of specific probe and screening
In line with conservative between type, belong between special principle, according to the comparison result of AlignX program, at a series of different lengths of the conserved regions design of pathogenic agent target gene (18 ~ 45nt) specific oligonucleotide probe.3 ' end of every bar probe adds 12 base T as connecting arm, and at 3 ' end T with amido modified, so that probe and chip carrier coupling.Use RT-PCR product that 16 kinds of pathogen nucleic acids are template and alternate probe to hybridize respectively, investigate each probe specificity, 16 specific probes (as shown in table 2) are determined in final screening.
Four, the preparation of gene chip
The centrifuge tube that probe (dry powder) is housed is placed on whizzer with the centrifugal 5min of 12000 turns/min, adding deionized water dissolving to concentration is 100 μMs, 10 μ l and chip sampling liquid (6 × SSC are got during point sample, 5% glycerine, 0.1%SDS) equal-volume mixing makes probe final concentration be 50 μMs.With commercially available gene chip sample applying instrument, in contact point sample mode, by probe points, to the aldehyde radicalization modification slide of blank, (chip schematic appearance is shown in accompanying drawing 1, array is as shown in table 3 with accompanying drawing 2), wherein Quality Control probe is 20T sequence, and blank is sampling liquid.Point process processed keep certain humidity, after preparation, chip should at least be placed 24 hours in drying at room temperature, make probe and carrier full cross-linked.
Five, the extraction of pathogen nucleic acid
Use the whole blood DNA/RNA rapid extraction test kit of QIAampMinEluteVirusSpinKit or the Mokogene company of Qiagen company to carry out the extraction of pathogen nucleic acid, operation by specification requires to carry out.
Six, RT-PCR amplification
The present invention adopts three groups of asymmetric multiple RT-PCR systems to carry out target gene amplification, and adopt the PrimeScriptOneStepRT-PCRKitVer.2 reagent of Takara company after condition optimizing, reaction system and amplification condition are in table 4.
Seven, chip hybridization
Amplified production is placed in 95 DEG C of sex change 5min, is placed in ice-water bath immediately and leaves standstill 5min, be placed in pre-washing lotion (0.2%SDS) before gene chip hybridization respectively and clean 20s with deionized water, dry for subsequent use.By hybridization solution (8 × SSC, 10% methane amide, 1.2%SDS, 10 × Denhardt ' s) heats and dissolves completely in 45 DEG C, three groups of amplified productions are respectively got and are added to chip hybridization reaction zone after 2 μ l and 6 μ l hybridization solutions mix and launch to spread even, and chip is placed in hybridizing box and hybridizes 1 hour in 45 DEG C.After hybridization terminates, take out chip, by its washing 20s in washing lotion A (1 × SSC, 0.2%SDS), washing lotion B (0.2 × SSC) and washing lotion C (0.1 × SSC) successively, dry rear preparation and develop the color.
Eight, the foundation of signal detecting method
Diluted with 1: 1000 by marking fluid (Streptavidin-HRP) diluent (1 × PBS+0.1%BSA), add 15 μ l in each hybridization region, chip is placed in 37 DEG C and hatches 30min.Clean 20s by PBST washing lotion (1 × PBS+0.05%Tween-20) after taking out, dry.By chemiluminescent HRP substrate A liquid and the mixing of B liquid (Millipore company) equal-volume, each hybridization district adds 20 μ l and expansion paving is even rapidly, is placed in chemiluminescence imaging instrument immediately and scans.Record result, judges result according to strength of signal.
Embodiment 2: hemorrhagic fever related diseases substance sldh gene chip Evaluation on specificity
Specificity is the most important performance assessment criteria of diagnostic method, and the present invention uses the system and condition optimized, and have detected 16 kinds of hemorrhagic fever related diseases pathogen nucleic acid, chip detection the results are shown in accompanying drawing 3.Result shows, utilizes the present invention 16 kinds of hemorrhagic fever related diseases substances correctly can be distinguished, has good specificity.
Embodiment 3: hemorrhagic fever related diseases substance sldh gene chip sensitivity evaluation
Use the detection sensitivity of sensitivity reference material to chip to evaluate, found that chip all can detect at least 10 to often kind of pathogenic agent 3rNA or DNA of copies/ system.For Ebola virus, plasmodium and heating companion thrombocytopenic syndromes virus, select 10 4copies/ μ l, 10 3copies/ μ l, 10 2copies/ μ l, 10 1pathogen nucleic acid and the negative control of copies/ μ l carry out chip detection, the results are shown in accompanying drawing 4.Result shows, chip all can detect at least 10 to often kind of pathogenic agent 3the pathogen nucleic acid of copies/ system, the detection sensitivity of chip is 10 2~ 10 3copies/ system.
In addition to the implementation, the present invention also has other embodiments, and every employing is equal to the technical scheme of replacement or equivalent transformation formation, all in the protection domain of application claims.

Claims (8)

1. a hemorrhagic fever related diseases substance sldh gene chip, can be used for detection 16 kinds of hemorrhagic fever related diseases pathogenic microorganisms (Zaire type Ebola virus, the Sudan type Ebola virus, Marburg virus, lassa virus, the peaceful virus of turtledove, Machupo virus, Rift Valley fever virus, crimean-Congo hemorrhagic fever virus, plasmodium, Hantaan virus, heating companion thrombocytopenic syndromes virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A, Type B influenza virus).It is characterized in that comprising 16 specific oligonucleotide probes detecting blood-head related diseases substance, 1 Quality Control probe, 29 Auele Specific Primers and carrier, each probe is fixed on carrier with latticed form.
2. gene chip according to claim 1, wherein said specific oligonucleotide probe is from the sequence listed by table 1.
Table 1 specific probe sequence
3. Quality Control probe according to claim 1, its sequence is 20T, and 5 ' end biotin labeling, 3 ' Amino End Group is modified.
4. gene chip according to claim 1, it is characterized in that comprising 16 kinds of hemorrhagic fever related diseases pathogenic microorganism specificity detection probe in array, its probe array is as shown in table 2 simultaneously.
Table 2 oligonucleotide probe array
Quality Control Quality Control Quality Control Quality Control Quality Control Quality Control Quality Control Quality Control ZEBO-P SEBO-P MAR-P LF-P JUN-P MAC-P RVF-P CCHFV-P ZEBO-P SEBO-P MAR-P LF-P JUN-P MAC-P RVF-P CCHFV-P ZEBO-P SEBO-P MAR-P LF-P JUN-P MAC-P RVF-P CCHFV-P MAL-P HV-MP SFTSV-P DEN-P YFV-P CHIKV-P FluA-P FluB-P MAL-P HV-MP SFTSV-P DEN-P YFV-P CHIKV-P FluA-P FluB-P MAL-P HV-MP SFTSV-P DEN-P YFV-P CHIKV-P FluA-P FluB-P Blank Blank Blank Blank Blank Blank Blank Blank
5. gene chip according to claim 1, is characterized in that described carrier can be the sheet glass or silicon chip, polystyrene substrate, nylon substrates modified through aldehyde radical or amination.
6. gene chip according to claim 1, is characterized in that the combination of primers comprising a kind of multiple hemorrhagic fever related diseases pathogen nucleic acid that simultaneously increases, comprises 29 Auele Specific Primers, and point three body systems increase.
7. combination of primers according to claim 5, wherein said primer sequence is from the sequence listed by table 3 or its combination.
Table 3 specific primer sequence
8. gene chip according to claim 1, its preparation method comprises the following steps.
Step one: prepare 29 pathogenic micro-organism primers (sequence is as shown in table 3), downstream primer 5 ' holds mark vitamin H, puts into 3 pipe RT-PCR systems respectively;
Step 2: prepare 16 specific probes (sequence is as shown in table 1), 3 ' end of every bar probe adds 12 base T and at 3 ' end with amido modified;
Step 3: by each oligonucleotide probe in step 2 and Quality Control probe chip sampling liquid (6 × SSC, 5% glycerine, 0.1%SDS) be diluted to final concentration 50 μMs, adopt contact point sample mode by probe points to (array is as shown in table 2) on carrier, point process processed keeps certain humidity, the point sample amount of all probes is 3nl, and after some system completes, chip at least should place 24 hours in drying at room temperature, make probe and carrier full cross-linked;
Step 4: fill a prescription as table 4 and prepare RT-PCR system:
Table 4RT-PCR system formulation
Step 5: prepare the pre-washing lotion of chip, its component is 0.2%SDS; Prepare hybridization solution, its component is 8 × SSC, 10% methane amide, 1.2%SDS, 10 × Denhardt ' s; Preparation washing lotion A, its component is 1 × SSC, 0.2%SDS; Preparation washing lotion B, its component is 0.2 × SSC; Preparation washing lotion C, its component is 0.1 × SSC;
Step 6: preparation detection reagent, comprises marking fluid (Streptavidin-HRP), diluent (1 × PBS+0.1%BSA), PBST washing lotion (1 × PBS+0.05%Tween-20), chemiluminescent HRP substrate A liquid and B liquid (Millipore company).
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CN109338016A (en) * 2018-11-06 2019-02-15 南通国际旅行卫生保健门诊部 Lassa virus rapid fluorescence PCR detection kit and its primer combination of probe
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CN110042173A (en) * 2019-02-28 2019-07-23 中国科学院广州生物医药与健康研究院 Nano-gold biosensor and detection method a kind of while that detect four kinds of hemorrhagic fever viruses
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
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CN105907888A (en) * 2016-04-14 2016-08-31 中国人民解放军军事医学科学院放射与辐射医学研究所 Making method and use of gene chip for detecting nine rash and fever pathogens
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CN110042173A (en) * 2019-02-28 2019-07-23 中国科学院广州生物医药与健康研究院 Nano-gold biosensor and detection method a kind of while that detect four kinds of hemorrhagic fever viruses
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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
CN114540551A (en) * 2022-03-14 2022-05-27 海南医学院 Liquid phase chip and method for simultaneously detecting three pathogens
CN114540551B (en) * 2022-03-14 2024-04-05 海南医学院 Liquid phase chip and method for simultaneously detecting three types of pathogens
CN115932238A (en) * 2022-08-05 2023-04-07 南京鼓楼医院 Probe and test strip for SFTS virus nucleic acid detection and use method
CN116987826A (en) * 2023-09-28 2023-11-03 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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