CN105483232A - Detection method for Rickettsia liquid phase gene chip - Google Patents
Detection method for Rickettsia liquid phase gene chip Download PDFInfo
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Abstract
The invention discloses a detection method for a Rickettsia liquid phase gene chip, relating to the field of gene chip detection, and being a quick and accurate pathogen detection technology with high flux. Multi-PCR is adopted to be combined with a liquid phase gene chip technology; a product amplified by multi-PCR is detected by the liquid phase gene chip technology, thus a defect that fragments similar in molecular weight cannot be differentiated by electrophoresis detection on the multi-PCR product, primer design freedom is greatly improved, and PCR reactions of a plurality of pairs of primers and even tens of pairs of primers in the same reaction tube become possible. In a detection process by the liquid phase gene chip technology, a biotin-avidin amplification system is adopted to be connected with fluorescent report molecules, and a chip fluorescence detector amplifies a reaction signal, so the detection sensitivity is greatly improved; the technology can be popularized and applied in departments of disease control, health quarantine and the like in China, and a quick, accurate and efficient detection means is provided, and plays an important role in the aspects of ensuring the health of people, dealing with sudden public health events, protecting public security and the like.
Description
Technical field
The present invention relates to genechip detection field, relate to a kind of detection method of rickettsia liquid phase gene chip in particular.
Background technology
Liquid-phase chip (liquidarray, liquidchip) also referred to as suspending chip (suspensionarray), complete multi-functional multi objective Synchronization Analysis system (FlexibleMultipleAnalyTE damping fluid Profiling by name, xMap), this technology utilizes the microballoon with unique encodings feature as reaction member, flow cytometer is as detection platform, measure on a large scale the biomolecules such as nucleic acid, protein, detection, screening all complete fast with being separated on same microballoon.Its organic combination laser technology, micro-fluidic technologies, fast signal process and data analysis system, both there is the feature that general sheet base biochip high-throughput detects, have again reproducible, highly sensitive, accuracy is good, be easy to stdn, the particular advantages such as qualitative and quantitative analysis simultaneously, has been widely used in the aspect such as pathogen detection, cytokines measurement at present.
Liquid-phase chip utilizes microballoon to react in the solution, overcome solid phase chip when macromole detects by surface tension, steric effect is isokinetic affects, greatly strengthen susceptibility, improve reaction efficiency; Utilize laser measuring technology simultaneously, substantially increase accuracy and the repeatability of sample detection, make it both have the high-throughout characteristic of solid phase chip in the past, there is again features such as being better than the easy and simple to handle, reproducible, highly sensitive of solid phase chip and detection by quantitative.Multiplex PCR adds multipair primer and to increase many target DNA fragments simultaneously in same reaction system, can detect multiple pathogenic microorganisms simultaneously, have the advantages such as efficient, quick.Multiplex PCR requires the specific amplification carrying out multiple site in same reaction system, and object fragment is separated by the general mode by agarose gel electrophoresis at present.Therefore, multiplex PCR also has many inevitable shortcomings, and as cross interference between different primers, between different primers, annealing temperature can not differ too large, the fragment length of amplification can not too close to etc., higher requirement is proposed to the design of primer.
The detection sensitivity of liquid-phase chip depends primarily on multiplexed PCR amplification efficiency and hybridization efficiency.By adjustment PCR reaction system and reaction conditions, amplification efficiency can be made to reach a preferably level.Specific fragment selection is the key of multiplexed PCR amplification efficiency and specific amplification.Hybridization efficiency is then relevant with the factor such as length, the sequence length of probe, the GC content of probe of amplified production.In liquid phase reaction, though hybridization conditions is identical, amplified production length, probe length, GC content are different, and cause the kinetics of each hybridization different, therefore the detection sensitivity of each probe is also variant.Although complete complementary pairing hybridization can be made to reach as far as possible at utmost by regulating the factor such as hybridization temperature, time, thus make all probes all reach a higher resolving power, but concerning corresponding multiple target sequence, be difficult to ensure that several hybridization all has enough rigors and sensitivity.
Summary of the invention
The invention provides a kind of detection method of rickettsia liquid phase gene chip, the multiple PCR primer amplification of selecting, fragment is all less than 100bp, complete the primer designed according to the PRIMER DESIGN STRATEGY of real-time fluorescence PCR, such amplification efficiency of target gene that can make improves greatly, thus adds the susceptibility of method.
For solving above-mentioned technical problem, the present invention by the following technical solutions:
A detection method for rickettsia liquid phase gene chip, comprises the steps:
(1) PCR reaction: 1. design primer, select the hot rickettsia of Q distinctive transposase htpAB gene-correlation tumor-necrosis factor glycoproteins, Rickettsia prowazeki condensing enzyme gltA gene and Rickettsia mooseri condensing enzyme gltA gene design primer respectively, analyze dimeric formation between each primer annealing temperature value and each primer by primer-design software; 2. PCR system and reaction conditions, PCR system (50 μ L) comprises PCR premixed liquid 25 μ L, each 1.0 μ L, the DNA3 μ L of upstream and downstream primer (10 μMs), and deionized water supplies 50 μ L; Reaction conditions: 94 DEG C of denaturations 5 minutes, 94 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations, last 72 DEG C extend 5 minutes;
(2) prepare, hybridize and detect microballoon, 5 ' end of every bar probe connects 20 poly base T as coupling end, and amination is modified;
1. connect probe and carboxylated microspheres, its step is as follows:
A, by dividing the carbodiimide powder installed from-20 DEG C of taking-ups, rise again to room temperature;
B, the oligonucleotide probe that amination is modified is resuspended in distilled water and makes its final concentration be 1mmol/L;
C, 1mL is packed in little spheroidal vibration 30 seconds, ultrasonic 30 seconds, disperse to bead;
D, get 100 μ L beads to 1.5mL centrifuge tube, 14000g4 minute, careful sucking-off supernatant;
E, microballoon are resuspended in 2-(N-morpholine) the ethyl sulfonic acid solution of 50 μ LpH4.5,0.1mol/L, vibrate 20 seconds, ultrasonic 20 seconds;
F, with the oligonucleotide probe 10 times dilution of distilled water by 1mmol/L;
G, the probe that 2 μ L10 doubly dilute is added in bead suspension, vibration mixing;
H, preparation 10mg/mL carbodiimide solution;
The freshly prepared carbodiimide solution of 2.5 μ L is added, vibration mixing in I, each 1.5ml centrifuge tube;
J, put on eddy oscillating device, room temperature lucifuge hatches 30 minutes;
The freshly prepared carbodiimide solution of 2.5 μ L is added, vibration mixing in K, often pipe;
L, again put on eddy oscillating device, room temperature lucifuge hatches 30 minutes;
After M, reaction terminate, centrifugal 4 minutes of 14000g, careful suction abandons supernatant;
Add 1mL0.02% polysorbas20 in N, each pipe and wash 1 time, polysorbas20 is a kind of nonionic surface active agent, contributes to macromole and dissolves;
O, centrifugal 14000g4 minute, careful suction abandons supernatant;
Add the sodium dodecyl sulfate solution of 1mL0.1% in P, each pipe, wash 1 time, supernatant is abandoned in the same centrifugal suction;
Add the TE damping fluid of 100 μ LpH8.0 in Q, each pipe, vibration 10s, ultrasonic 10s, make microballoon resuspended;
The counting of R, microballoon: count with blood counting chamber;
S, the microballoon having connect probe are suspended from the TE damping fluid of pH8.0, and 4 DEG C save backup;
2. connect the Quality Control of microballoon, its step is as follows:
A, synthesis and the oligonucleotide chain of probes complementary, oligonucleotide chain 5 ' end biotin labeling;
B, fully vibrate with eddy oscillating device the coding microball connect;
C, draw appropriate microballoon, be diluted to 3500/25 μ L with 2 × tetramethylammonium chloride hybridization solution, be placed in 0.2 μ L centrifuge tube;
D, dilution and oligonucleotide chain to the 0.1 μm ol/L of probes complementary;
The above-mentioned oligonucleotide solution of 10 μ L is added and 15 μ LTE buffer solns make its final volume be 50 μ L, piping and druming mixing in E, each pipe;
F, 95 DEG C of sex change 10 minutes;
G, 55 DEG C hybridization 15 minutes;
H, be transferred to filter plate suction filtration and remove unconjugated nucleotide chain;
I, each hole add 1 × tetramethylammonium chloride hybridization solution liquid of 75 μ l4ng/ μ L streptavidin-phycoerythrin, and room temperature lucifuge hatches 10 minutes, and suction filtration removes unconjugated streptavidin-phycoerythrin;
J, each hole add 75 μ L1 × tetramethylammonium chloride hybridization solution solution, and vibration makes microballoon resuspended;
K, reaction terminate rear use liquid phase suspension chip instrument and detect;
L, arrange and do not add oligonucleotide chain and the negative control only adding TE damping fluid simultaneously, the samely to detect;
Result judges: the MFI value of the microballoon of connection is greater than 5000, illustrates that bag is by success;
3. hybridization and detection, its step is as follows:
A, get each 3500 of the microballoon having connect probe and be mixed in 25 μ L2 × tetramethylammonium chloride hybridization solution hybridization solution, be placed in 0.2 μ L centrifuge tube;
Add PCR primer and the 15 μ LTE buffer solns of 10 μ L hybrid templates in B, each pipe, make its final volume be 50 μ L, piping and druming mixing;
C, 95 DEG C of sex change 10 minutes;
Certain hour is hybridized under D, hybridization temperature;
E, be transferred to filter plate suction filtration and remove unconjugated PCR primer;
F, each hole add 1 × tetramethylammonium chloride hybridization solution liquid of 75 μ L4ng/ μ L streptavidin-phycoerythrin, and room temperature lucifuge hatches 10 minutes, and suction filtration removes unconjugated streptavidin-phycoerythrin;
G, each hole add 75 μ L1 × tetramethylammonium chloride hybridization solution solution, and vibration makes microballoon resuspended, terminate rear use liquid phase suspension chip instrument and detect;
H, by operation instructions, first flushing pipe, then preheating laser detector 30 minutes, stand-by;
I, according to microballoon numbering and the factor to be checked, write detect control;
J, quantity according to sample, set up operating process;
K, then 96 orifice plates are put into detect storehouse detect;
During detection, carry out hybridization check as detection background using PCR negative control simultaneously, for each detection system and detection background, the data that instrument exports are average fluorescent strength values of a kind of numbering population of microspheres in respective reaction system, be MFI value, that is the statistical average value of each microballoon strength of signal of the population of microspheres of this numbering read, each hole MFI value and background fluorescence value is read by liquid phase suspension chip instrument suspension chip system, background fluorescence value is BFI value, in conjunction with liquid phase suspension chip instrument analyzing and processing data; When the MFI value of sample to be detected is this detection background strength of signal more than three times, be namely judged to the positive;
(3) multi-primers liquid phase genechip detection system is set up: mixing is linked with 3 kinds of microballoon composition Multiple detection systems of 3 Species specific probes respectively;
(4) PCR primer is detected: every hole adds 25 μ L and contains 2 × tetramethylammonium chloride hybridization solution hybridization solution of each 3500 of microballoon, 10 μ LPCR products and 15 μ LTE buffer solns in Multiple detection system;
(5) preparation, hybridization and the hybridization conditions detected in microballoon are optimized:
optimize hybridization temperature, hybridization temperature selects 47 DEG C, 51 DEG C, 55 DEG C, 59 DEG C, 63 DEG C 5 temperature to carry out respectively, with the PCR primer of hybrid template for detected material, carries out result judgement with MFI value and the ratio of BFI value;
optimize hybridization time, after determining hybridization temperature, with the PCR primer of hybrid template for detected material, hybridization time gets 10 minutes, 15 minutes, 20 minutes, 25 minutes respectively, more different hybridization time on the impact of strength of signal,
(6) Method validation of liquid phase gene chip: 1. specificity verification, selects multiple bacterial strain, examines multiple suspension chip method to the specificity of sample detection; 2. sensitivity checking, the plasmid of different concns increases according to the PCR system after optimizing respectively, is then detected by the microballoon of corresponding probe with wrapping, and liquid phase suspension chip instrument system carries software and carries out analysis and draw dose response curve; BFI value is signal when negative control detects, and lowest detectable limit estimates from matched curve, and namely the MFI value of analyte is equivalent to the detection substrate concentration corresponding to BFI value three times; 3. repeatability checking, is template with plasmid, after pcr amplification, uses the microballoon with a collection of dressing probe, detects carrying out repeatability with a collection of PCR primer; 4. simulated samples checking, not carry rickettsial mouse body sample as experiment pattern, by the mouse body sample adding plasmid and do not add plasmid mouse body sample in contrast, extraction DNA detects simultaneously.
Compared with prior art, the invention has the beneficial effects as follows:
(1) the hot rickettsia of Q, Rickettsia prowazeki, Rickettsia mooseri specific detection primer and probe is devised;
(2) establish the liquid phase gene chip detection method of the hot rickettsia of Q, Rickettsia prowazeki, Rickettsia mooseri, and demonstrate the sensitivity of method, specificity and repeatability by plasmid system, analog sample and other pathogenic agent known.
Accompanying drawing explanation
Fig. 1 is the hot rickettsia of Q and Rickettsia prowazeki specificity verification result electrophorogram;
Fig. 2 is Rickettsia mooseri specificity verification result electrophorogram;
Fig. 3 is the result of multiplex PCR system;
Fig. 4 is annealing temperature optimum result electrophorogram;
Fig. 5 is primer pair concentration optimization result electrophorogram;
Fig. 6 is the optimum result figure of hybridization temperature;
Fig. 7 is the optimum result figure of hybridization time;
Fig. 8 is three kinds of probe liquid phase gene chip specificity verification result figure;
Fig. 9 is three kinds of rickettsia liquid phase gene chip specificity verification result figure;
Figure 10 is the hot rickettsia DNA concentration of Q-MFI value dose response curve figure;
Figure 11 is Rickettsia prowazeki DNA concentration-MFI value dose response curve figure;
Figure 12 is Rickettsia mooseri DNA concentration-MFI value dose response curve figure;
Figure 13 is three kinds of rickettsia liquid phase gene chip simulated samples the result figure.
Embodiment
Application principle of the present invention, effect and effect, be explained by following embodiment.
Embodiment 1
The detection method of a kind of rickettsia liquid phase gene chip provided by the invention, comprises the steps:
(1) PCR reaction: 1. design primer, select the hot rickettsia of Q distinctive transposase htpAB gene-correlation tumor-necrosis factor glycoproteins, Rickettsia prowazeki condensing enzyme gltA gene and Rickettsia mooseri condensing enzyme gltA gene design primer respectively, analyze dimeric formation between each primer annealing temperature value and each primer by primer-design software; 2. PCR system and reaction conditions, PCR system (50 μ L) comprises PCR premixed liquid 25 μ L, each 1.0 μ L, the DNA3 μ L of upstream and downstream primer (10 μMs), and deionized water supplies 50 μ L; Reaction conditions: 94 DEG C of denaturations 5 minutes, 94 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations, last 72 DEG C extend 5 minutes;
(2) prepare, hybridize and detect microballoon, 5 ' end of every bar probe connects 20 poly base T as coupling end, and amination is modified;
1. connect probe and carboxylated microspheres, its step is as follows:
A, by dividing the carbodiimide powder installed from-20 DEG C of taking-ups, rise again to room temperature;
B, the oligonucleotide probe that amination is modified is resuspended in distilled water and makes its final concentration be 1mmol/L;
C, 1mL is packed in little spheroidal vibration 30 seconds, ultrasonic 30 seconds, disperse to bead;
D, get 100 μ L beads to 1.5mL centrifuge tube, 14000g4 minute, careful sucking-off supernatant;
E, microballoon are resuspended in 2-(N-morpholine) the ethyl sulfonic acid solution of 50 μ LpH4.5,0.1mol/L, vibrate 20 seconds, ultrasonic 20 seconds;
F, with the oligonucleotide probe 10 times dilution of distilled water by 1mmol/L;
G, the probe that 2 μ L10 doubly dilute is added in bead suspension, vibration mixing;
H, preparation 10mg/mL carbodiimide solution;
The freshly prepared carbodiimide solution of 2.5 μ L is added, vibration mixing in I, each 1.5ml centrifuge tube;
J, put on eddy oscillating device, room temperature lucifuge hatches 30 minutes;
The freshly prepared carbodiimide solution of 2.5 μ L is added, vibration mixing in K, often pipe;
L, again put on eddy oscillating device, room temperature lucifuge hatches 30 minutes;
After M, reaction terminate, centrifugal 4 minutes of 14000g, careful suction abandons supernatant;
Add 1mL0.02% polysorbas20 in N, each pipe and wash 1 time;
O, centrifugal 14000g4 minute, careful suction abandons supernatant;
Add the sodium dodecyl sulfate solution of 1mL0.1% in P, each pipe, wash 1 time, supernatant is abandoned in the same centrifugal suction;
Add the TE damping fluid of 100 μ LpH8.0 in Q, each pipe, vibration 10s, ultrasonic 10s, make microballoon resuspended;
The counting of R, microballoon: count with blood counting chamber;
S, the microballoon having connect probe are suspended from the TE damping fluid of pH8.0, and 4 DEG C save backup;
2. connect the Quality Control of microballoon, its step is as follows:
A, synthesis and the oligonucleotide chain of probes complementary, oligonucleotide chain 5 ' end biotin labeling;
B, fully vibrate with eddy oscillating device the coding microball connect;
C, draw appropriate microballoon, be diluted to 3500/25 μ L with 2 × tetramethylammonium chloride hybridization solution, be placed in 0.2 μ L centrifuge tube;
D, dilution and oligonucleotide chain to the 0.1 μm ol/L of probes complementary;
The above-mentioned oligonucleotide solution of 10 μ L is added and 15 μ LTE buffer solns make its final volume be 50 μ L, piping and druming mixing in E, each pipe;
F, 95 DEG C of sex change 10 minutes;
G, 55 DEG C hybridization 15 minutes;
H, be transferred to filter plate suction filtration and remove unconjugated nucleotide chain;
I, each hole add 1 × tetramethylammonium chloride hybridization solution liquid of 75 μ l4ng/ μ L streptavidin-phycoerythrin, and room temperature lucifuge hatches 10 minutes, and suction filtration removes unconjugated streptavidin-phycoerythrin;
J, each hole add 75 μ L1 × tetramethylammonium chloride hybridization solution solution, and vibration makes microballoon resuspended;
K, reaction terminate rear use liquid phase suspension chip instrument and detect;
L, arrange and do not add oligonucleotide chain and the negative control only adding TE damping fluid simultaneously, the samely to detect;
Result judges: the MFI value of the microballoon of connection is greater than 5000, illustrates that bag is by success;
3. hybridization and detection, its step is as follows:
A, get each 3500 of the microballoon having connect probe and be mixed in 25 μ L2 × tetramethylammonium chloride hybridization solution hybridization solution, be placed in 0.2 μ L centrifuge tube;
Add PCR primer and the 15 μ LTE buffer solns of 10 μ L hybrid templates in B, each pipe, make its final volume be 50 μ L, piping and druming mixing;
C, 95 DEG C of sex change 10 minutes;
Certain hour is hybridized under D, hybridization temperature;
E, be transferred to filter plate suction filtration and remove unconjugated PCR primer;
F, each hole add 1 × tetramethylammonium chloride hybridization solution liquid of 75 μ L4ng/ μ L streptavidin-phycoerythrin, and room temperature lucifuge hatches 10 minutes, and suction filtration removes unconjugated streptavidin-phycoerythrin;
G, each hole add 75 μ L1 × tetramethylammonium chloride hybridization solution solution, and vibration makes microballoon resuspended, terminate rear use liquid phase suspension chip instrument and detect;
H, by operation instructions, first flushing pipe, then preheating laser detector 30 minutes, stand-by;
I, according to microballoon numbering and the factor to be checked, write detect control;
J, quantity according to sample, set up operating process;
K, then 96 orifice plates are put into detect storehouse detect;
During detection, carry out hybridization check as detection background using PCR negative control simultaneously, for each detection system and detection background, the data that instrument exports are average fluorescent strength values of a kind of numbering population of microspheres in respective reaction system, be MFI value, that is the statistical average value of each microballoon strength of signal of the population of microspheres of this numbering read, each hole MFI value and background fluorescence value is read by liquid phase suspension chip instrument suspension chip system, background fluorescence value is BFI value, in conjunction with liquid phase suspension chip instrument analyzing and processing data; When the MFI value of sample to be detected is this detection background strength of signal more than three times, be namely judged to the positive;
(3) multi-primers liquid phase genechip detection system is set up: mixing is linked with 3 kinds of microballoon composition Multiple detection systems of 3 Species specific probes respectively;
(4) PCR primer is detected: every hole adds 25 μ L and contains 2 × tetramethylammonium chloride hybridization solution hybridization solution of each 3500 of microballoon, 10 μ LPCR products and 15 μ LTE buffer solns in Multiple detection system;
(5) preparation, hybridization and the hybridization conditions detected in microballoon are optimized:
optimize hybridization temperature, hybridization temperature selects 47 DEG C, 51 DEG C, 55 DEG C, 59 DEG C, 63 DEG C 5 temperature to carry out respectively, with the PCR primer of hybrid template for detected material, carries out result judgement with MFI value and the ratio of BFI value;
optimize hybridization time, after determining hybridization temperature, with the PCR primer of hybrid template for detected material, hybridization time gets 10 minutes, 15 minutes, 20 minutes, 25 minutes respectively, and more different hybridization time is on the impact of strength of signal
(6) Method validation of liquid phase gene chip: 1. specificity verification, selects multiple bacterial strain, examines multiple suspension chip method to the specificity of sample detection; 2. sensitivity checking, the plasmid of different concns increases according to the PCR system after optimizing respectively, is then detected by the microballoon of corresponding probe with wrapping, and liquid phase suspension chip instrument (same as above) system carries software and carries out analysis and draw dose response curve; BFI value is signal when negative control detects, and lowest detectable limit estimates from matched curve, and namely the MFI value of analyte is equivalent to the detection substrate concentration corresponding to BFI value three times; 3. repeatability checking, is template with plasmid, after pcr amplification, uses the microballoon with a collection of dressing probe, detects carrying out repeatability with a collection of PCR primer; 4. simulated samples checking, not carry rickettsial mouse body sample as experiment pattern, by the mouse body sample adding plasmid and do not add plasmid mouse body sample in contrast, extraction DNA detects simultaneously.
In the present embodiment, detected result is as follows:
One, without optimization means
As Fig. 1, shown in Fig. 2, in Fig. 1,1-6 is respectively the hot rickettsia of Q hot rickettsia primer pair Q, Rickettsia prowazeki, Rickettsia mooseri, francisella tularensis, Yersinia pestis, the amplification of negative control, 7-12 is respectively Rickettsia prowazeki, the hot rickettsia of Q, Rickettsia mooseri, francisella tularensis, Yersinia pestis, the amplification of negative control, in Fig. 2,1-6 is respectively Rickettsia mooseri primer pair Rickettsia mooseri, the hot rickettsia of Q, Rickettsia prowazeki, francisella tularensis, Yersinia pestis, the amplification of negative control, by single amplification to primer, amplified band is there is in the template that each pair of primer is all corresponding to it in corresponding position, pillar location is correct, and control strain amplification does not all have bands visible.
As shown in Figure 3, in figure, 1-5 is respectively the amplification of the hot rickettsia+Rickettsia prowazeki+Rickettsia mooseri template of Q, the hot rickettsia of Q, Rickettsia prowazeki, Rickettsia mooseri, negative control, three pairs of primer mixing amplifications, all there is amplified band in corresponding position in each template, pillar location is correct.Because fragment length is too close, hybrid template and single template PCR primer cannot be distinguished by electrophoresis.
Two, to after PCR condition optimizing:
1. annealing temperature is optimized, annealing temperature is respectively the amplification of 53 DEG C, 55 DEG C, 57 DEG C as shown in Figure 4, in figure, 1-3 is respectively annealing temperature 53 DEG C, the hot rickettsia amplification of the Q of 55 DEG C, 57 DEG C, 4-6 is respectively annealing temperature 53 DEG C, the Rickettsia prowazeki of 55 DEG C, 57 DEG C, and 7-9 is respectively annealing temperature 53 DEG C, the Rickettsia mooseri of 55 DEG C, 57 DEG C; As seen from the figure, when the hot rickettsia annealing temperature of Q is 55 DEG C, amplification better; Rickettsia prowazeki annealing temperature be 53 DEG C and 55 DEG C time, amplification is better; Rickettsia mooseri annealing temperature is that 53 DEG C, 55 DEG C and 57 DEG C are all better, does not have notable difference.Therefore, 55 DEG C are selected as the annealing temperature of triple PCR amplification.
2. primer concentration is optimized, primary structure is revised according to the annealing temperature after optimizing, carry out primer concentration optimization experiment, upstream and downstream primer (10 μMs) volume is respectively 2.5 μ L, 2.0 μ L, 1.5 μ L, the amplification of 1.0 μ L as shown in Figure 5, in figure, 1-4 is respectively each 2.5 μ L of Q hot rickettsia upstream and downstream primer (10 μMs), 2.0 μ L, 1.5 μ L, the amplification of 1.0 μ L, 5-8 is respectively the same amplification of Rickettsia prowazeki, 9-12 is respectively the same amplification of Rickettsia mooseri, as seen from Figure 5, along with each 2.5 μ L of upstream and downstream primer (10 μMs) volume, 2.0 μ L, 1.5 μ L, the change of 1.0 μ L, the hot rickettsia of Q, Rickettsia prowazeki and Rickettsia mooseri amplified production amount do not have considerable change, and the amount of Rickettsia prowazeki and Rickettsia mooseri primer dimer when upstream and downstream primer concentration increases also increases.Therefore, select each 1.0 μ L of upstream and downstream primer (10 μMs) volume as consumption.
Therefore, the present invention PCR reaction system is set and reaction conditions as follows:
PCR system (50 μ L) comprises premixed liquid, 25 μ L; The each 1.0 μ L of upstream and downstream primer (10 μMs); DNA3 μ L; Deionized water supplies 50 μ L.
Reaction conditions: 94 DEG C of denaturations 5 minutes; 94 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations; Last 72 DEG C extend 5 minutes.
Three, after hybridization conditions being optimized
optimize hybridization temperature, as shown in Figure 6, each probe signal to noise ratio change differ, but 63 DEG C and 67 DEG C of signal to noise ratios substantially on a declining curve; Integrated comparative signal to noise ratio at different temperatures, and consider the impact of temperature on hybrid specificities, select hybridization temperature to be 59 DEG C;
optimize hybridization time, as shown in Figure 7, along with the prolongation of hybridization time, each probe signal to noise ratio increases substantially thereupon, therefore selects hybridization time to be 25 minutes.
To sum up, hybridization underlying condition is optimized for 95 DEG C of sex change 10min, then hybridizes 25 minutes for 59 DEG C.
Four, the Method validation result of liquid phase gene chip
specificity verification, the specificity verification of probe, namely with alternate probe, factor various combination to be checked is detected, when there being a certain factor to be checked to exist, the corresponding microballoon of this detecting factor shows strong fluorescent signal, and all the other microballoons do not have considerable change compared with the fluorescent signal on negative Quality Control microballoon, illustrate to there is not cross reaction between each probe and other analytes, as shown in Figure 8, this system has the ability detecting the hot rickettsia of Q, Rickettsia prowazeki and Rickettsia mooseri; As shown in Figure 9, specific amplification is occurred to the hot rickettsia of Q, Rickettsia prowazeki, Rickettsia mooseri, and there is not amplification in francisella tularensis, Yersinia pestis EV76 strain
In sum, this detection technique possesses good specificity.
2. sensitivity checking, as shown in Figure 10, Q hot rickettsia liquid phase suspension chip instrument analytical results draws matched curve, and curvilinear equation is:
FI=-6.53652+ (1881.23+6.53652)/((1+ (Conc/14.9405)
-1.61702))
0.916998, detect from typical curve reckoning and be limited to 1.5 × 10
3copies/mL;
As shown in Figure 11, Rickettsia prowazeki liquid phase suspension chip instrument analytical results draws matched curve, and curvilinear equation is:
FI=8.93662+ (2781.56-8.93662)/((1+ (Conc/0.0795255)
-1.13837))
10, detect from typical curve reckoning and be limited to 3.9 × 10
3copies/mL;
As shown in Figure 12, Rickettsia mooseri liquid phase suspension chip instrument analytical results draws matched curve, and curvilinear equation is:
FI=4.94624+ (2842.66-4.94624)/((1+ (Conc/0.590861)
-0.623733))
10, detect from typical curve reckoning and be limited to 3.5 × 10
3copies/mL;
In sum, this detection technique possesses stronger sensitivity.
3. repeatability checking, observe the MFI value detected for 4 times, the hot rickettsia of Q, Rickettsia prowazeki and the Rickettsia mooseri variation coefficient are respectively 3.28%, 12.62% and the hot rickettsia of 2.78%, Q and Rickettsia mooseri repeatability better, and Rickettsia prowazeki repeatability is more weak.
4. simulated samples checking, as shown in Figure 13, adds in the mouse body sample of plasmid and detect the hot rickettsia of Q, Rickettsia prowazeki, Rickettsia mooseri, and the mouse body sample not adding plasmid does not detect.
Be embodiments of the invention as mentioned above.The present invention is not limited to above-mentioned embodiment, and anyone should learn the structural changes made under enlightenment of the present invention, and every have identical or close technical scheme with the present invention, all falls within protection scope of the present invention.
Claims (1)
1. a detection method for rickettsia liquid phase gene chip, is characterized in that, comprises the steps:
(1) PCR reaction: 1. design primer, select the hot rickettsia of Q distinctive transposase htpAB gene-correlation tumor-necrosis factor glycoproteins, Rickettsia prowazeki condensing enzyme gltA gene and Rickettsia mooseri condensing enzyme gltA gene design primer respectively, analyze dimeric formation between each primer annealing temperature value and each primer by primer-design software; 2. PCR system and reaction conditions, PCR system (50 μ L) comprises PCR premixed liquid 25 μ L, each 1.0 μ L, the DNA3 μ L of upstream and downstream primer (10 μMs), and deionized water supplies 50 μ L; Reaction conditions: 94 DEG C of denaturations 5 minutes, 94 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations, last 72 DEG C extend 5 minutes;
(2) prepare, hybridize and detect microballoon, 5 ' end of every bar probe connects 20 poly base T as coupling end, and amination is modified;
1. connect probe and carboxylated microspheres, its step is as follows:
A, by dividing the carbodiimide powder installed from-20 DEG C of taking-ups, rise again to room temperature;
B, the oligonucleotide probe that amination is modified is resuspended in distilled water and makes its final concentration be 1mmol/L;
C, 1mL is packed in little spheroidal vibration 30 seconds, ultrasonic 30 seconds, disperse to bead;
D, get 100 μ L beads to 1.5mL centrifuge tube, 14000g4 minute, careful sucking-off supernatant;
E, microballoon are resuspended in 2-(N-morpholine) the ethyl sulfonic acid solution of 50 μ LpH4.5,0.1mol/L, vibrate 20 seconds, ultrasonic 20 seconds;
F, with the oligonucleotide probe 10 times dilution of distilled water by 1mmol/L;
G, the probe that 2 μ L10 doubly dilute is added in bead suspension, vibration mixing;
H, preparation 10mg/mL carbodiimide solution;
The freshly prepared carbodiimide solution of 2.5 μ L is added, vibration mixing in I, each 1.5ml centrifuge tube;
J, put on eddy oscillating device, room temperature lucifuge hatches 30 minutes;
The freshly prepared carbodiimide solution of 2.5 μ L is added, vibration mixing in K, often pipe;
L, again put on eddy oscillating device, room temperature lucifuge hatches 30 minutes;
After M, reaction terminate, centrifugal 4 minutes of 14000g, careful suction abandons supernatant;
Add 1mL0.02% polysorbas20 in N, each pipe and wash 1 time;
O, centrifugal 14000g4 minute, careful suction abandons supernatant;
Add the sodium dodecyl sulfate solution of 1mL0.1% in P, each pipe, wash 1 time, supernatant is abandoned in the same centrifugal suction;
Add the TE damping fluid of 100 μ LpH8.0 in Q, each pipe, vibration 10s, ultrasonic 10s, make microballoon resuspended;
The counting of R, microballoon: count with blood counting chamber;
S, the microballoon having connect probe are suspended from the TE damping fluid of pH8.0, and 4 DEG C save backup;
2. connect the Quality Control of microballoon, its step is as follows:
A, synthesis and the oligonucleotide chain of probes complementary, oligonucleotide chain 5 ' end biotin labeling;
B, fully vibrate with eddy oscillating device the coding microball connect;
C, draw appropriate microballoon, be diluted to 3500/25 μ L with 2 × tetramethylammonium chloride hybridization solution, be placed in 0.2 μ L centrifuge tube;
D, dilution and oligonucleotide chain to the 0.1 μm ol/L of probes complementary;
The above-mentioned oligonucleotide solution of 10 μ L is added and 15 μ LTE buffer solns make its final volume be 50 μ L, piping and druming mixing in E, each pipe;
F, 95 DEG C of sex change 10 minutes;
G, 55 DEG C hybridization 15 minutes;
H, be transferred to filter plate suction filtration and remove unconjugated nucleotide chain;
I, each hole add 1 × tetramethylammonium chloride hybridization solution liquid of 75 μ l4ng/ μ L streptavidin-phycoerythrin, and room temperature lucifuge hatches 10 minutes, and suction filtration removes unconjugated streptavidin-phycoerythrin;
J, each hole add 75 μ L1 × tetramethylammonium chloride hybridization solution solution, and vibration makes microballoon resuspended;
K, reaction terminate rear use liquid phase suspension chip instrument and detect;
L, arrange and do not add oligonucleotide chain and the negative control only adding TE damping fluid simultaneously, the samely to detect;
Result judges: the MFI value of the microballoon of connection is greater than 5000, illustrates that bag is by success;
3. hybridization and detection, its step is as follows:
A, get each 3500 of the microballoon having connect probe and be mixed in 25 μ L2 × tetramethylammonium chloride hybridization solution hybridization solution, be placed in 0.2 μ L centrifuge tube;
Add PCR primer and the 15 μ LTE buffer solns of 10 μ L hybrid templates in B, each pipe, make its final volume be 50 μ L, piping and druming mixing;
C, 95 DEG C of sex change 10 minutes;
Certain hour is hybridized under D, hybridization temperature;
E, be transferred to filter plate suction filtration and remove unconjugated PCR primer;
F, each hole add 1 × tetramethylammonium chloride hybridization solution liquid of 75 μ L4ng/ μ L streptavidin-phycoerythrin, and room temperature lucifuge hatches 10 minutes, and suction filtration removes unconjugated streptavidin-phycoerythrin;
G, each hole add 75 μ L1 × tetramethylammonium chloride hybridization solution solution, and vibration makes microballoon resuspended, terminate rear use liquid phase suspension chip instrument and detect;
H, by operation instructions, first flushing pipe, then preheating laser detector 30 minutes, stand-by;
I, according to microballoon numbering and the factor to be checked, write detect control;
J, quantity according to sample, set up operating process;
K, then 96 orifice plates are put into detect storehouse detect;
During detection, carry out hybridization check as detection background using PCR negative control simultaneously, for each detection system and detection background, the data that instrument exports are average fluorescent strength values of a kind of numbering population of microspheres in respective reaction system, be MFI value, that is the statistical average value of each microballoon strength of signal of the population of microspheres of this numbering read, each hole MFI value and background fluorescence value is read by liquid phase suspension chip instrument suspension chip system, background fluorescence value is BFI value, in conjunction with liquid phase suspension chip instrument analyzing and processing data; When the MFI value of sample to be detected is this detection background strength of signal more than three times, be namely judged to the positive;
(3) multi-primers liquid phase genechip detection system is set up: mixing is linked with 3 kinds of microballoon composition Multiple detection systems of 3 Species specific probes respectively;
(4) PCR primer is detected: every hole adds 25 μ L and contains 2 × tetramethylammonium chloride hybridization solution hybridization solution of each 3500 of microballoon, 10 μ LPCR products and 15 μ LTE buffer solns in Multiple detection system;
(5) preparation, hybridization and the hybridization conditions detected in microballoon are optimized:
optimize hybridization temperature, hybridization temperature selects 47 DEG C, 51 DEG C, 55 DEG C, 59 DEG C, 63 DEG C 5 temperature to carry out respectively, with the PCR primer of hybrid template for detected material, carries out result judgement with MFI value and the ratio of BFI value;
optimize hybridization time, after determining hybridization temperature, with the PCR primer of hybrid template for detected material, hybridization time gets 10 minutes, 15 minutes, 20 minutes, 25 minutes respectively, and more different hybridization time is on the impact of strength of signal;
(6) Method validation of liquid phase gene chip: 1. specificity verification, selects multiple bacterial strain, examines multiple suspension chip method to the specificity of sample detection; 2. sensitivity checking, the plasmid of different concns increases according to the PCR system after optimizing respectively, is then detected by the microballoon of corresponding probe with wrapping, and liquid phase suspension chip instrument system carries software and carries out analysis and draw dose response curve; BFI value is signal when negative control detects, and lowest detectable limit estimates from matched curve, and namely the MFI value of analyte is equivalent to the detection substrate concentration corresponding to BFI value three times; 3. repeatability checking, is template with plasmid, after pcr amplification, uses the microballoon with a collection of dressing probe, detects carrying out repeatability with a collection of PCR primer; 4. simulated samples checking, not carry rickettsial mouse body sample as experiment pattern, by the mouse body sample adding plasmid and do not add plasmid mouse body sample in contrast, extraction DNA detects simultaneously.
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