CN104651498A - Preparation and application of rickettsia discrimination detection gene chip - Google Patents

Preparation and application of rickettsia discrimination detection gene chip Download PDF

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CN104651498A
CN104651498A CN201510033678.4A CN201510033678A CN104651498A CN 104651498 A CN104651498 A CN 104651498A CN 201510033678 A CN201510033678 A CN 201510033678A CN 104651498 A CN104651498 A CN 104651498A
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rickettsia
chip
probe
gene
gene chip
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王升启
李灵云
刘琪琦
陈苏红
张敏丽
张英杰
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Institute of Radiation Medicine of CAMMS
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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 preparation and application of a rickettsia discrimination detection gene chip. The preparation method comprises the following steps: preparing universal and specific primers, preparing seven rickettsia nucleic acid typing probes, preparing an oligonucleotide chip, establishing a multiplex PCR (polymerase chain reaction) system, and establishing a hybridization system. The gene chip can simultaneously discriminate seven important rickettsiae, including rickettsia prowazeki, rickettsia mooseri, spotted fever group rickettsia, Coxiella burnetii, Orientia tsutsugamushi, Ehrlichia chaffeensis and Anaplasma phagocytophilum. The gene chip has the advantages of high speed, high accuracy, high flux and high specificity, and can provide a novel detection means for clinical diagnosis and epidemiological investigation of rickettsia infection.

Description

Rickettsia screens the preparation and purposes that detect gene chip
Technical field
The present invention relates to rickettsia and screen the preparation and purposes that detect gene chip, belong to gene chip detecting technique field.
Background technology
Rickettsia (rickettsiae) is the entozoic Gram-negative bacteria of class special sexual cell except only a few, its rickettsiosis caused is the disease of natural focus of the important infecting both domestic animals and human of a class, worldwide in sporadic and seasonal epidemic.Many arthropodss such as lice, flea, tick, mite etc. all can be used as the communication media of rickettsial infection.Along with the development of international tourism, rickettsiosis is considered to one of Important Infectious Diseases threatening tourism healthy.In the last few years, the continuous appearance of Spotted Fever novel species, it is popular that Ehrlichia etc. were newly sent out rickettsial, all reminding our the rickettsiosiss popular possibility with breaking out on a large scale.In addition rickettsia is as potential bio-terrorism pathogenic agent, also day by day comes into one's own, and the U.S. opposes that epidemic typhus, Rocky Mountains spotted fever and Q heat are listed in biological warfare agent register by bioterrorism (Bioterrorism).Therefore, rickettsial monitoring is strengthened and control has important practical significance.
Found in current world wide that having pathogenic rickettsia has more than 20 to plant, the isolated pathogenic agent of patient and gene test evidence show that China at least exists 10 kinds of rickettsiosiss.Rickettsiosis early symptom is various, and without specific clinical feature, rate of clinical misdiagnosis is high.Delay treatment can cause serious complication, even dead.But if correct diagnosis can be made in early days in infection, cheap, effective doxycycline and tsiklomitsin can be effective to rickettsia.Therefore early diagnosis is fast and accurately the key controlling rickettsiosis.Traditional rickettsia detection method mainly contains separation and Culture, PCR and immunology diagnosis.But separation and Culture length consuming time, complicated operation; PCR and immunological method can only detect one or more pathogenic agent, can not meet the rickettsial needs detecting wide variety completely.And biochip technology has fast, accurately, the feature such as low cost is specially adapted to the high throughput testing of rickettsiosis.
Summary of the invention
The object of the invention is to the some shortcomings existed for rickettsia nucleic acid high throughput testing field, develop a kind of high-throughput, special, responsive, the detection of rickettsia examination fast gene chip, seven kinds of important rickettsia can be detected simultaneously, comprise Rickettsia prowazeki, Rickettsia mooseri, Fever Group Rickettsiae, Coxiella burnetii, Orientia Tsutsugamushi, Ehrlichia chaffeensis, Anaplasma phagocytophila, for the clinical diagnosis of rickettsial infection and epidemiology survey provide a kind of new detection means.
In order to achieve the above object, rickettsia of the present invention is screened and is detected gene chip, and its preparation method is as follows:
1. step one: prepare the general and Auele Specific Primer of rickettsiae
Selecting the ompB gene, Coxiella burnetii icd gene, Orientia Tsutsugamushi 47-KDa gene, Anaplasma phagocytophila ankA gene, Ehrlichia chaffeensis trp-120 gene etc. of rickettsiae as detecting target gene, the amplification of 7 kinds of rickettsia target fragments can be realized.The preferably amplification target of 6 pairs of primer sequences and correspondence thereof, as shown in table 1:
The amplification target of table 1 primer sequence and correspondence
2. step 2: prepare specific oligonucleotide probe
According to the sequence alignment in the comparison between 7 kinds of rickettsia gene orders and often kind of rickettsia kind, the relatively special district of the sequence within the scope of upstream and downstream primer carries out the design of typing probes.The target of rickettsia general probe sequence, Species specific probes sequence and correspondence, as shown in table 2:
The target of table 2 sequence oligonucleotide probe and correspondence
3. step 3: prepare oligonucleotide chip
A preferred embodiment, each oligonucleotide probe in step 2, when point sample, is diluted to final concentration 50 μMs with 2 × sampling liquid (6 × SSC, 0.1%SDS).The aldehyde radicalization of probe points to blank modified on slide with commercially available gene chip sample applying instrument, the point sample amount of probe is 3nl.After oligonucleotide chip preparation, before using, at least place dry 18 hours in room temperature.This chip features is to comprise rickettsiae general probe and 7 Species specific probes in oligonucleotide probe array simultaneously, and its probe array is as shown in table 3.Wherein sheet matrix control probe is 20T sequence, and 5 ' end bio mark, 3 ' end NH2 modify, and is used for monitoring aldehyde radical blade matrix amount; Negative probes is the plant genetic sequences irrelevant with rickettsia, is used to refer to specificity; The point sample amount of all probes is 3nl, at least places dry 18 hours in room temperature before using.
Table 3 oligonucleotide probe array
The control of sheet matrix The control of sheet matrix The control of sheet matrix The control of sheet matrix The control of sheet matrix The control of sheet matrix
The control of sheet matrix RP RT SFGR-a SFGR-b R
The control of sheet matrix RP RT SFGR-a SFGR-b R
The control of sheet matrix RP RT SFGR-a SFGR-b R
The control of sheet matrix Q-P Ot-P EC-P AP-P NC
The control of sheet matrix Q-P Ot-P EC-P AP-P NC
The control of sheet matrix Q-P Ot-P EC-P AP-P NC
4. step 4: set up multiple asymmetric PCR system
In gene chip of the present invention, the feature of PCR system is the triple asymmetric PCR reaction system of 2 pipe.Suitable multiple asymmetric PCR system can improve the sensitivity of chip detection further.The factors such as the consumption of the absolute concentration of labeled primer and non-marked primer and relative proportion, Taq enzyme are optimized, preferred multiplex PCR system, as shown in table 4:
The multiple asymmetric PCR system formulation of table 4
Preferred pcr amplification condition is: 95 DEG C of denaturation 2min; 95 DEG C of 20s, 55 DEG C of 20s, 72 DEG C, 20s, totally 45 circulations; 72 DEG C extend 5min.
5. step 5: set up hybridization system
Suitable hybridization system also has great role to the specificity of chip and sensitivity improving.By optimizing the hybridization solution composition, hybridization conditions and the post-hybridization wash conditions that obtain and can ensure specificity and sensitivity simultaneously.In hybridization system, PCR primer (A pipe and B manage each 2.5 μ l) mixes with hybridization solution (5 μ l) equal-volume, and each composition final concentration of preferred hybridization solution is 4 × SSC, 0.3%SDS, 5% methane amide.Preferred hybridization conditions is that 45 DEG C of water-baths hybridize 1 hour.Preferred wash conditions is washing lotion A under normal temperature (1 × SSC, 0.2%SDS), respectively washs 20s in washing lotion B (0.2 × SSC) and washing lotion C (0.1 × SSC).
6. step 6: chemoluminescence develops the color
1) 10 μ l marking fluids are added in chip reaction zone---horseradish peroxidase-labeled streptavidin (streptavidin-HRP), 30min is placed in 37 DEG C of water-baths; Clean 20s by PBST washing lotion (1 × PBS+0.05%Tween20) after taking out, repeat 3 times, put room temperature and dry.2) by colouring reagents A liquid and the mixing of B liquid equal-volume, each chip reaction zone immediately lucifuge adds A, B mixed solution of 20 μ l, gene chip is put into the imaging of portable chemical luminous organism chip imager, the chip hybridization signal of collection uses ArrayVision7.0 software analysis result.
The rickettsia more than prepared is screened and is detected gene chip, comprises oligonucleotide chip, PCR system, hybridization solution, washing lotion A, washing lotion B, washing lotion C, marking fluid, nitrite ion A, nitrite ion B.
A preferred embodiment uses commercially available DNA extraction kit to extract rickettsia genomic dna, extracts and carries out with reference to corresponding test kit specification sheets.The DNA solution extracted uses multiplex PCR system to increase according to the amplification condition in step 4.PCR primer mixes with hybridization solution equal-volume, adds in oligonucleotide chip, carries out hybridizing and developing the color according to the condition of step 5 and step 6.
Chip after washing uses portable chemical luminous organism chip imager to scan, and operation analysis software sentence read result.Select the bacteriums such as mycoplasma pneumoniae, Chlamydia pneumoniae, streptococcus aureus, pseudomonas cepacia, streptococcus pneumoniae, Pseudomonas aeruginosa, hemophilus influenzae, Klebsiella Pneumoniae and stenotrophomonas maltophilia as sample, utilize the gene chip of above-mentioned preparation to detect, investigate the specificity of chip.Preparation plasmid DNA sensitivity reference material, investigates the lowest detectable limit of chip.Result: Rickettsia mooseri, Spotted fever Group Ricketfsiase detect and be limited to 3 × 50copies/ reaction, Rickettsia prowazeki, Ehrlichia chaffeensis detect and are limited to 3 × 100copies/ reaction, and Orientia Tsutsugamushi, Coxiella burnetii, Anaplasma phagocytophila detect and be limited to 3 × 1000copies/ reaction.
Use genechip detection rickettsia analog sample (whole blood) prepared by the present invention.This chip method detects the rickettsia analog sample of 4 kinds of different concns, and result shows, and analog sample genechip detection coincidence rate reaches 100%, and sensitivity is 10 3-10 4copies/ μ l.Show that the method for gene chip that this experiment is set up accurately can detect the rickettsia in whole blood.
The present invention establishes a kind of gene chip based on chemiluminescence imaging method, seven kinds of important rickettsia can be screened simultaneously, comprise Rickettsia prowazeki, Rickettsia mooseri, Fever Group Rickettsiae, Coxiella burnetii, Orientia Tsutsugamushi, Ehrlichia chaffeensis, Anaplasma phagocytophila.The advantage that this gene chip has fast, accurate, high-throughput, specificity are high, the clinical diagnosis and the epidemiology survey that can be rickettsial infection provide a kind of new detection means.Performance shows, gene chip of the present invention is accurately to screen 7 kinds of rickettsia not of the same race, and specificity is good.Chip of the present invention is 150-3000copies/ reaction to 7 kinds of rickettsial detection sensitivities.
Accompanying drawing explanation
Fig. 1: for rickettsia of the present invention screens the array schematic diagram detecting gene chip, often open on chip and be distributed with 10 identical arrays.
Fig. 2: concrete alignment placement on each array on rickettsia examination detection gene chip.In figure, initial point represents a point sample of probe, 3 round dots of vertical direction be a probe repeat point samples 3 times.1. corresponding is Rickettsia prowazeki specific probe; 2. corresponding is Rickettsia mooseri specific probe; 3. corresponding is Fever Group Rickettsiae specific probe 1; 4. corresponding is Fever Group Rickettsiae specific probe 2; 5. corresponding is the universal probe of rickettsiae; 6. corresponding is Coxiella burnetii specific probe; 7. corresponding is Orientia Tsutsugamushi specific probe; 8. corresponding is Ehrlichia chaffeensis specific probe; 9. corresponding is Anaplasma phagocytophila specific probe; 10. corresponding is negative control probe; counterpiece matrix control probe.
Fig. 3: typical 7 kinds of rickettsial chip detection figure.Wherein 1-7 is followed successively by Rickettsia prowazeki, Rickettsia mooseri, Rana amurensis, Ehrlichia chaffeensis, Coxiella burnetii, Orientia Tsutsugamushi, Anaplasma phagocytophila rickettsia examination chip detection result.
Fig. 4: gene chip specific detection result figure.Wherein 1-10 is followed successively by the detected result figure of mycoplasma pneumoniae, Chlamydia pneumoniae, streptococcus aureus, pseudomonas cepacia, streptococcus pneumoniae, Pseudomonas aeruginosa, C type hemophilus influenzae, Type B hemophilus influenzae, stenotrophomonas maltophilia, Klebsiella Pneumoniae.
The sensitivity technique result figure of Fig. 5: 7 kinds of rickettsia plasmid DNA reference materials.Wherein 1-7 is followed successively by Rickettsia prowazeki 3 × 10 5copies, 3 × 10 4copies, 3 × 10 3copies, 3 × 10 2copies, 3 × 50copies, 3 × 10 1copies plasmid DNA reference material and negative control detected result; 8-14 is followed successively by Rickettsia mooseri 3 × 10 5copies, 3 × 10 4copies, 3 × 10 3copies, 3 × 10 2copies, 3 × 50copies, 3 × 10 1copies plasmid DNA reference material and negative control detected result; 15-21 is followed successively by Rana amurensis 3 × 10 5copies, 3 × 10 4copies, 3 × 10 3copies, 3 × 10 2copies, 3 × 50copies, 3 × 10 1copies plasmid DNA reference material and negative control detected result; 22-28 is followed successively by Ehrlichia chaffeensis 3 × 10 5copies, 3 × 10 4copies, 3 × 10 3copies, 3 × 10 2copies, 3 × 50copies, 3 × 10 1copies plasmid DNA reference material and negative control detected result; 29-36 is followed successively by Coxiella burnetii 3 × 10 5copies, 3 × 10 4copies, 3 × 10 3copies, 3 × 500copies, 3 × 10 2copies, 3 × 10 1copies plasmid DNA reference material and negative control detected result; 37-42 is followed successively by Orientia Tsutsugamushi 3 × 10 5copies, 3 × 10 4copies, 3 × 10 3copies, 3 × 500copies, 3 × 10 2copies, 3 × 10 1copies plasmid DNA reference material and negative control detected result; 43-48 is followed successively by Anaplasma phagocytophila 3 × 10 5copies, 3 × 10 4copies, 3 × 10 3copies, 3 × 500copies, 3 × 10 2copies, 3 × 10 1copies plasmid DNA reference material and negative control detected result.
Fig. 6: rickettsia is screened and detected gene chip analog sample detected result figure.1-5 is followed successively by Rickettsia prowazeki 10 5copies/ μ l, 10 4copies/ μ l, 10 3copies/ μ l, 10 2copies/ μ l analog sample and negative sample detected result; 6-10 is followed successively by Rickettsia mooseri 10 5copies/ μ l, 10 4copies/ μ l, 10 3copies/ μ i, 10 2copies/ μ l analog sample and negative sample detected result; 11-15 is followed successively by Rana amurensis 10 5copies/ μ l, 10 4copies/ μ l, 10 3copies/ μ l, 10 2copies/ μ l analog sample and negative sample detected result; 16-20 is followed successively by Ehrlichia chaffeensis 10 5copies/ μ l, 10 4copies/ μ l, 10 3copies/ μ l, 10 2copies/ μ l analog sample and negative sample detected result; 21-25 is followed successively by Coxiella burnetii 10 5copies/ μ l, 10 4copies/ μ l, 10 3copies/ μ l, 10 2copies/ μ l analog sample and negative sample detected result; 29-30 is followed successively by Orientia Tsutsugamushi 10 5copies/ μ l, 10 4copies/ μ l, 10 3copies/ μ l, 10 2copies/ μ l analog sample and negative sample detected result; 31-35 is followed successively by Anaplasma phagocytophila 10 5copies/ μ l, 10 4copies/ μ l, 10 3copies/ μ l, 10 2copies/ μ l analog sample and negative sample detected result.
Embodiment
The following example is intended to illustrate instead of limit the present invention.
Embodiment 1: rickettsia screens the development detecting gene chip
One, primed probe design and screening
First from NCBI gene database, rickettsia target-gene sequence is downloaded, after sequence has been downloaded, the AlignX program in Vector NTIAdvance 10 (invitrogen) software package is used to carry out overall comparison according to the optimum configurations of acquiescence to each pathogen gene sequence.According to comparison result at the conservative Position Design specific oligonucleotide probe of gene order, general and Auele Specific Primer.Finally determine totally 12 upstream and downstream primers through screening, reverse primer is carried out 5 ' end bio mark, as the primer that chip uses; Determine 10 specificity detection probe, 3 ' end NH 2modify.
Two, oligonucleotide chip preparation and probe array
After completing probe screening, determine final probe array, see accompanying drawing 1 and accompanying drawing 2.1. corresponding is Rickettsia prowazeki specific probe; 2. corresponding is Rickettsia mooseri specific probe; 3. corresponding is Fever Group Rickettsiae specific probe 1; 4. corresponding is Rickettsia spoted fever group specific probe 2; 5. corresponding is the universal probe of rickettsiae; 6. corresponding is Coxiella burnetii specific probe; 7. corresponding is Orientia Tsutsugamushi specific probe; 8. corresponding is Ehrlichia chaffeensis specific probe; 9. corresponding is Anaplasma phagocytophila specific probe; 10. corresponding is negative control probe; counterpiece matrix control probe.
Three, multiple asymmetric PCR system
In the present invention, the feature of PCR system is the triple asymmetric PCR system of two pipes.Suitable PCR system can improve the sensitivity of chip detection further.The factors such as the consumption of the absolute concentration of labeled primer and non-marked primer and relative proportion, Taq enzyme are optimized.When upstream and downstream primer final concentration is 0.1 μM: 0.5 μM, when Taq enzyme consumption is 2.5U/ system, the probe signals value of reference material is comparatively strong, and low copy template 10 3copie/ μ l still can detect.Preferred pcr amplification condition is: 95 DEG C of denaturation 2min; 95 DEG C of 20s, 55 DEG C of 20s, 72 DEG C, 20s, totally 45 circulations; 72 DEG C extend 5min.
Four, set up and optimize hybridization system
By optimizing the hybridization solution composition, hybridization conditions and the post-hybridization wash conditions that obtain ensureing specificity and sensitivity simultaneously.In hybridization system, PCR primer mixes with hybridization solution equal-volume, and each composition final concentration of hybridization solution is 4 × SSC, 0.3%SDS, 5% methane amide.Hybridization conditions is that 45 DEG C of water-baths hybridize 1 hour.Wash conditions is washing lotion A under normal temperature (1 × SSC, 0.2%SDS), respectively washs 20s in washing lotion B (0.2 × SSC) and washing lotion C (0.1 × SSC).
Five, chemoluminescence colour developing
1) 10 μ l marking fluids are added in chip reaction zone---horseradish peroxidase-labeled streptavidin (streptavidin-HRP), 30min is placed in 37 DEG C of water-baths; Clean 20s by PBST washing lotion (1 × PBS+0.05%Tween20) after taking out, repeat 3 times, put room temperature and dry.
2) by colouring reagents A liquid and the mixing of B liquid equal-volume, each chip reaction zone immediately lucifuge adds A, B mixed solution of 20 μ l, gene chip is put into the imaging of portable chemical luminous organism chip imager, the chip hybridization signal of collection uses ArrayVision7.0 software analysis result.
Embodiment 2: rickettsia screens the determination detecting gene chip Positive judgement standards
Cutoff value judges whether gene chip signal value is positive standard.Every bar typing probes chooses non-rickettsia (i.e. negative strain) respectively, blank carries out gene chip hybridization, by experiment repeatedly and data statistics, using the Cutoff value of the Background statistic mean value+2SD of negative strain and blank as every bar probe.Whether the separating capacity of each abrupt climatic change probe be there is more than 2.5 times the judging criterion of suddenling change as site.
Embodiment 3: rickettsia screens the Evaluation on specificity detecting gene chip
Specificity is the most important performance assessment criteria of diagnostic method, gene chip of the present invention uses the system and condition optimized, have detected the bacteriums such as mycoplasma pneumoniae, Chlamydia pneumoniae, streptococcus aureus, pseudomonas cepacia, streptococcus pneumoniae, Pseudomonas aeruginosa, hemophilus influenzae, Klebsiella Pneumoniae and stenotrophomonas maltophilia, as can be seen from accompanying drawing 4, above-mentioned Bacteria Detection result is all that rickettsia is negative, and specificity is good.The present invention detects 7 kinds of rickettsia, and as can be seen from accompanying drawing 3,7 kinds of rickettsia all can obviously be distinguished, and illustrate that specificity of the present invention is good.
Embodiment 4: rickettsia is screened and detected gene chip sensitivity evaluation
Build 7 kinds of rickettsia plasmid DNA as detection reference material, from 10 9copies/ μ l gradient dilution to 10 1copies/ μ l, carries out chip detection, the results are shown in accompanying drawing 5.As can be seen from accompanying drawing 5, detectability of the present invention: Rickettsia mooseri, Spotted fever Group Ricketfsiase detect and be limited to 3 × 50copies/ reaction; Rickettsia prowazeki, Ehrlichia chaffeensis detect and are limited to 3 × 100copies/ reaction; Orientia Tsutsugamushi, Coxiella burnetii, Anaplasma phagocytophila detect and are limited to 3 × 1000copies/ reaction.
Embodiment 5: rickettsia is screened and detected the detection of gene chip analog sample
Use gene chip prepared by the present invention, detect the rickettsia analog sample (whole blood) of 4 kinds of different concns, result shows, and analog sample genechip detection coincidence rate reaches 100%, and sensitivity is 10 3-10 4copies/ μ l.Partial detection is shown in accompanying drawing 6.
In addition to the implementation, the present invention also has other embodiments.Every employing is equal to the technical scheme of replacement or equivalent transformation formation, all at the protection domain of application claims.

Claims (10)

1. screen detection seven kinds of rickettsial gene chips, gene chip of the present invention may be used for screening Rickettsia prowazeki, Rickettsia mooseri, Fever Group Rickettsiae, Coxiella burnetii, Orientia Tsutsugamushi, Ehrlichia chaffeensis, Anaplasma phagocytophila simultaneously.It is characterized in that comprising and detect rickettsial 12 general and Auele Specific Primers; Article 8, rickettsia specific oligonucleotide probe; Article 1, rickettsiae general probe; 1 silver matrix control probe and 1 negative control probe and carrier.Above-mentioned probe is distributed on carrier respectively.
Table 1 rickettsiae universal amplification primer
Table 2 Coxiella burnetii amplimer
Table 3 Orientia Tsutsugamushi amplimer
Table 4 Anaplasma phagocytophila amplimer
Table 5 Ehrlichia chaffeensis amplimer
Table 6 rickettsia specific oligonucleotide probe sequence
2. the rickettsia according to right 1 is screened and is detected gene chip, it is characterized in that described carrier is sheet glass, silicon chip, polystyrene substrate, the nylon substrates that aldehyde radicalization is modified.
3. rickettsia screens the preparation method detecting gene chip, comprises the following steps:
Step one, the design of probe and primer: first download rickettsia gene order from NCBI gene database, after sequence has been downloaded, the AlignX program in Vector NTI Advance 10 (invitrogen) software package is used to carry out overall comparison according to the optimum configurations of acquiescence to each pathogen gene sequence.According to conservative Position Design specific oligonucleotide probe, the Auele Specific Primer of comparison result in gene order.
Step 2, the synthesis of probe, 3 ' end of every bar probe adds 12 base T and 3 ' end T is amido modified as connecting arm, modifies on glass substrate can be fixed on aldehyde radicalization; Quality Control probe except 3 ' end T carry out amido modified except, 5 ' end with tense marker biotin labeling;
Step 3, the preparation of chip: the probe deionized water after synthesis is diluted to 100 μMs, get 10 μ L probe solutions respectively, with 10 μ L volume chip sampling liquid mixings, probe point sample final concentration is made to be 50 μMs, be loaded on 384 orifice plates, chip surface is sticked 10 sample well array films, with Pixsys 5000 chip preparing instrument (Cartesian Technologies), adopt contact point sample mode, by probe points on carrier, certain humidity is kept in deposition process, after point sample completes, chip is placed in moisture eliminator lucifuge normal temperature and leaves standstill 48h, probe and chip surface aldehyde radical is made to slough covalent attachment after 1 molecular water, the chip Air drying that point makes is preserved.
4. rickettsia according to claim 3 screens the preparation method detecting gene chip, and it is characterized in that 10 specific oligonucleotide probes and 12 primers in step one, probe length is at 20-37nt, and primer length is at 18-25nt.
5. rickettsia according to claim 3 screens the preparation method detecting gene chip, it is characterized in that the carrier described in step 3 kind is aldehyde radical glass chip or silicon chip, polystyrene substrate, nylon substrates.
6. rickettsia screens the using method detecting gene chip, it is characterized in that comprising the following steps:
1) step one, the extraction of rickettsia genomic dna, uses commercial goods bacterial genomes DNA extraction kit to extract rickettsia genomic dna;
2) step 2, multiple asymmetric PCR amplification: amplification uses the PCR reagent of TaKaRa company, is divided into A, B two pipe by whole amplification system.A pipe comprises Rickettsia mooseri, Rickettsia prowazeki, Fever Group Rickettsiae, Ehrlichia chaffeensis; B pipe comprises Coxiella burnetii, Orientia Tsutsugamushi, Anaplasma phagocytophila.Amplification is increased by following loop parameter: 95 DEG C of denaturation 2min; 95 DEG C of 20s, 55 DEG C of 20s, 72 DEG C, 20s, totally 45 circulations; 72 DEG C extend 5min; Preserve or carry out next step experiment for 4 DEG C;
3) step 3, chip hybridization: gene chip is set to 0 in .2%SDS and deionized water respectively and cleans 30s respectively, centrifugal drying; Amplified production step 2 obtained is placed in ice bath 5min immediately after 95 DEG C of sex change 5min; get the A pipe product 2.5 μ L of sex change and B pipe product 2.5 μ L and 5 μ L hybridization solutions mix; use sample injector to be added on chip well and makes its uniform fold in array surface, gene chip is put into the inherent 45 DEG C of hybridization 1h of hybridizing box;
4) step 4, post-hybridization washes gene chip: after gene chip hybridization completes, from hybridizing box, take out chip, and in washing lotion 1 × SSC+0.2%SDS, 0.2 × SSC and 0.1 × SSC, respectively clean 30S successively immediately, finally by gene chip surface liquid centrifugal drying;
5) step 5, sample marks: add 15 μ l marking fluids to chip, put back to by chip in the mid-37 DEG C of water-baths of hybridizing box react 30min with pipettor after smoothening, and takes out chip and cleans 10s with PBST, centrifugal drying;
6) step 6, scanning: the mixing solutions adding luminescent solution A and B of just 1: 1 mixing to chip reaction zone, puts immediately in chemiluminescence imaging instrument after smoothening scan with pipettor, imaging pattern is trigger mode, exposure parameter 511, gain parameter 300, time shutter 10s, triggering times 1 time;
7) step 6, data analysis: imaging terminates rear use chemiluminescence analysis software and carries out chip probe signal analysis.The signal of every bar probe gets its three mean values repeated a little, and according to probe Cutoff value, the interpretation of this probe of probe signals value > Cutoff value is that this probe signals is positive.The reverse primer decorating molecule that in the step 2 of using method of the present invention, PCR uses is vitamin H.The hybridization solution component used in using method step 3 of the present invention is 8 × SSC, 0.6%SDS, 10% methane amide, 10 × Denhardt.
7. rickettsia according to claim 6 screens the using method detecting gene chip, it is characterized in that the end of the reverse primer 5 ' for each specific gene of the rickettsia that increases used in step 2 is modified.
8. the terminal modified molecule of reverse primer 5 ' according to claim 7 can be CY3, CY5, vitamin H.
9. rickettsia according to claim 6 screens the using method detecting gene chip, it is characterized in that the hybridization solution component used in step 3 is 8 × SSC, 0.6%SDS, 10% methane amide, 10 × Denhardt.
10. rickettsia according to claim 6 screens the using method detecting gene chip, it is characterized in that the scan method used in step 6, the difference of reverse primer decorating molecule according to Claim 8, scan method comprises fluorescent scanning, visual scanning, chemiluminescence imaging.
CN201510033678.4A 2015-01-23 2015-01-23 Preparation and application of rickettsia discrimination detection gene chip Pending CN104651498A (en)

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CN104962628A (en) * 2015-06-25 2015-10-07 中华人民共和国北京出入境检验检疫局 Detection reagent box, primer and probe which are used for detecting spotted fever group rickettsias and rickettsia mooseri simultaneously
CN105018610A (en) * 2015-07-14 2015-11-04 宋锋林 Dual real-time fluorescence quantitative PCR (polymerase chain reaction) detection system and method for Rickettsia mooseri and Orientia tsutsugamushi
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Cited By (15)

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Publication number Priority date Publication date Assignee Title
CN104962628A (en) * 2015-06-25 2015-10-07 中华人民共和国北京出入境检验检疫局 Detection reagent box, primer and probe which are used for detecting spotted fever group rickettsias and rickettsia mooseri simultaneously
CN105018610B (en) * 2015-07-14 2019-02-15 宋锋林 Rickettsia mooseri and Orientia Tsutsugamushi dual real-time fluorescence quantitative PCR detection system and detection method
CN105018610A (en) * 2015-07-14 2015-11-04 宋锋林 Dual real-time fluorescence quantitative PCR (polymerase chain reaction) detection system and method for Rickettsia mooseri and Orientia tsutsugamushi
CN105255884A (en) * 2015-11-24 2016-01-20 吉林出入境检验检疫局检验检疫技术中心 Detection primer set of bainite rickettsia and detection reagent of detection primer set and real-time fluorescent quantitative PCR method
CN105255884B (en) * 2015-11-24 2018-05-04 吉林出入境检验检疫局检验检疫技术中心 The detection primer group and its detection reagent of Rickettsia belii, real time fluorescence quantifying PCR method
CN105543346A (en) * 2015-12-24 2016-05-04 四川国际旅行卫生保健中心 Multiplex fluorescence PCR (polymerase chain reaction) detection method for rickettsia
CN105543346B (en) * 2015-12-24 2019-04-16 四川国际旅行卫生保健中心 A kind of Richettsia multiple fluorescence PCR detection method
CN108531649A (en) * 2018-04-11 2018-09-14 四川农业大学 A kind of enzymatic of the synchronous detection diarrhoeal virus of 4 boars visualizes oligonucleotide chip and its application
CN108531649B (en) * 2018-04-11 2022-03-22 四川农业大学 Enzymatic visual oligonucleotide chip for synchronously detecting 4 porcine diarrheal viruses and application thereof
CN109355409A (en) * 2018-10-09 2019-02-19 中国农业大学 A kind of high-throughput visualization quickly chip of detection Richettsia and application
CN109355409B (en) * 2018-10-09 2022-01-25 中国农业大学 Chip for high-flux visual rapid detection of rickettsia and application
CN113265479A (en) * 2021-05-26 2021-08-17 中国人民解放军军事科学院军事医学研究院 Primer composition for detecting rickettsia morganii and application thereof
CN113444773A (en) * 2021-08-17 2021-09-28 黑龙江省牡丹江林业中心医院 Method and kit for detecting tick pathogen nucleic acid based on liquid chip
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

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