CN103114033A - Array type multiplex microfluidic isothermal amplification chip for HPV (human papillomavirus) detection - Google Patents
Array type multiplex microfluidic isothermal amplification chip for HPV (human papillomavirus) detection Download PDFInfo
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- CN103114033A CN103114033A CN2012104434886A CN201210443488A CN103114033A CN 103114033 A CN103114033 A CN 103114033A CN 2012104434886 A CN2012104434886 A CN 2012104434886A CN 201210443488 A CN201210443488 A CN 201210443488A CN 103114033 A CN103114033 A CN 103114033A
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Abstract
The invention discloses an array type multiplex microfluidic isothermal amplification chip for HPV (human papillomavirus) detection, and the chip is made from polydimethylsiloxane and is prepared in a pouring method by utilizing a micro-processing technology. The basic structure of the chip comprises three parts, namely amplification pools, connecting pipelines and capillary channels. A series of the amplification pools which are orderly arranged in a space array are coated with nucleic acid probes through a freeze-drying technology, and the simultaneous detection of multiplex nucleic acid targets in a sample can be achieved by utilizing space differentiation of amplification signals; the capillary channels can achieve the pouring of a reaction solution and the sample through capillary pulling force, and a low-mass transfer coefficient is simultaneously utilized for effectively preventing the mutual interference of the nucleic acid probes, amplification products and the signals in different amplification pools; and the amplification pools and the capillary channels are communicated through the connecting pipelines for enabling fluid to smoothly and uniformly flow into the amplification pools. The chip is simple to manufacture and convenient to operate, and provides an effective scheme for simultaneous clinical detection of a variety of pathogens through an LAMP (isothermal amplification) method.
Description
Technical field
The present invention relates to a kind of multiple nucleic acid isothermal amplification technology, relate in particular to the multiple microfluid constant-temperature amplification of a kind of array chip.
Background technology
Human papillomavirus HPV is that a kind of papilloma vacuolating virus A that belongs to papovaviridae belongs to, and is spherical DNA virus.Divide high-risk type and low risk, low risk HPV such as HPV-6,11,13,32,34,40,42,43,44,53,54 grades and infection sexual organ, anus, pars oralis pharyngis, esophageal mucosa, particularly HPV6,11, incidence is the most extensive.The persistent infection of high-risk human mammilla papillomavirus is the major cause that causes cervical cancer, and wherein HPV 16,18 types are with cervical cancer, 2 kinds of high-risk human mammilla papillomavirus the closest, that the risk of causing a disease is the highest to occur, and account at least more than 40% of cervical cancer.In time distinguish rapidly dissimilar important HPV and have vital effect for prevention and treatment of diseases, to the individual or society all is significant.
Ring mediated isothermal nucleic acid amplification (Loop-mediated isothermal amplification, LAMP) the earliest by Japanese scientist's invention, that this technology has is highly sensitive, specificity good, speed of response is fast, the direct advantage such as naked eyes judgement of reaction result, through the academic research of nearly 10 years, existing form with various detection kit came into the market." the isothermal pattern " but of LAMP reaction and its complicated nucleic acid probe make it be difficult to realize in same system detect in the plurality of target thing in a sample, i.e. Multiple detection.Microflow control technique is to control technology and the science that skin rises the volume fluid that rise to of receiving in the micro-meter scale structure, the laminar flow effect, surface tension effect, capillary effect, heat-conduction effect and the diffusional effect that significantly strengthen under this yardstick, make many physics and chemistry processes that the variation of matter occur, current just in one of booming new and high technology and Environment Science field, be the important technological platform of future of life science, chemistry and information science development, the application of this technology can realize the Multiple detection pattern of LAMP reaction.
Summary of the invention
The objective of the invention is for the deficiencies in the prior art, a kind of multiple microfluid constant-temperature amplification of array chip for the HPV detection is provided.
The object of the present invention is achieved like this: a kind of multiple microfluid constant-temperature amplification of array chip for high-risk HPV detection, it comprises capillary channel, connecting tube, well, some amplifications pond and some probe filling orifices, described well communicates with capillary channel, one end in all ponds of increasing all is communicated with capillary channel by connecting tube, and each is communicated with the other end with a probe filling orifice; Wherein, be equipped with positive probe in the pond of increasing, this positive probe has the DNA sequence dna shown in SEQ ID NO.25-SEQ ID NO.28, is equipped with negative probe in another pond of increasing, described negative probe is water, and described positive probe and negative probe are in order to improve the confidence level of detected result; Be coated with respectively upper specific probe for the target nucleic acid fragment in all the other ponds 1 of increasing; Described specific probe for the target nucleic acid fragment is by for HPV-6,11 the probe with the DNA sequence dna shown in SEQ ID NO.1-SEQ ID NO.12, for the probe with the DNA sequence dna shown in SEQ ID NO.13-SEQ ID NO.18 of HPV-16 with for one or more compositions in the probe with the DNA sequence dna shown in SEQ ID NO.19-SEQ ID NO.24 of HPV-18.
Further, described connecting tube is quadrangular frustum pyramid shaped, and the end that cross section is large is connected with the pond of increasing, and the end that cross section is little is connected with capillary channel, makes microfluid steadily flow into uniformly from capillary channel the pond of increasing, and does not produce bubble.Described amplification pool space size is according to its volume demand, and its length can change arbitrarily with demand, the general ￡ 1mm of the bulk of width and the degree of depth; The general ￡ 0.3mm of the width of capillary channel and the degree of depth can.
The invention has the beneficial effects as follows, the multiple microfluid constant-temperature amplification of array of the present invention chip can be applicable to merge fast the nucleic acid that detects HPV6 type and HPV11 type, and somatotype detects the nucleic acid of HPV16 type and HPV18 type.
Description of drawings
Fig. 1 is the structural representation of the multiple microfluid constant-temperature amplification of array chip;
In figure, pond 1, connecting tube 2, capillary channel 3, well 4, probe filling orifice 5 increase.
Embodiment
As shown in Figure 1, the multiple microfluid constant-temperature amplification of array of the present invention chip comprises capillary channel 3, well 4, some amplifications pond 1 and some probe filling orifices 5, well 4 communicates with capillary channel 3, one end in all ponds 1 of increasing all is communicated with capillary channel 3 by connecting tube 2, and each is communicated with the other end with a probe filling orifice 5.
Be equipped with positive probe in the pond 1 of increasing, this positive probe has the DNA sequence dna shown in SEQ ID NO.25-SEQ ID NO.28, be equipped with negative probe in the pond of increasing, described negative probe is water, and positive probe and negative probe are in order to improve the confidence level of detected result.Except the above-mentioned amplification pond that is equipped with respectively positive probe and negative probe 1, coated upper specific probe for the target nucleic acid fragment respectively in all the other amplifications ponds 1.As for HPV-6,11 the probe with the DNA sequence dna shown in SEQ ID NO.1-SEQ ID NO.12, for the probe with the DNA sequence dna shown in SEQ ID NO.13-SEQ ID NO.18 of HPV-16, for the probe with the DNA sequence dna shown in SEQ ID NO.19-SEQ ID NO.24 of HPV-18.The Demand Design that the quantity in amplification pond 1 can detect according to target compound is as triple, quadruple, five heavy or ten heavily wait, unrestricted.
Capillary channel 3 is mainly each amplification specific dna probe and the reaction product crossed contamination in different ponds in pond of characteristic limitations that utilizes its physics confinement (inferior quality transmission coefficient), also utilizes pulling force capillaceous to be convenient to the sample introduction of sample and reaction solution simultaneously.
Be communicated with connecting tube 2 between amplification pond 1 and capillary channel 3, connecting tube 2 is quadrangular frustum pyramid shaped, and the end that cross section is large is connected with the pond 1 of increasing, and the end that cross section is little is connected with capillary channel 3, make microfluid steadily flow into uniformly from capillary channel 3 pond 4 of increasing, and do not produce bubble.
Amplification pool space size is according to its volume demand, and its length can change arbitrarily with demand, the general ￡ 1mm of the bulk of width and the degree of depth; The general ￡ 0.3mm of the width of capillary channel and the degree of depth can; It is fixed that the bulk of connecting tube 2 is come according to the size of amplification pond 1 and capillary channel 3, mainly plays a kind of even connection effect.
The multiple microfluid constant-temperature amplification of array of the present invention chip prepares by following steps:
1, the making of chip template: utilize the method for micro-processing technology (MEMS, photoetching etc.) to make the chip template, formwork structure is corresponding with the multiple microfluid constant-temperature amplification of above-mentioned array chip, and mould material can be silicon materials.
2, chip cast, degassed and solidify: as after 5-12:1 mixes in mass ratio with dimethyl siloxane and solidifying agent, carefully to be poured on the chip template, after vacuum outgas 30 min 80
oC solidifies 2h, and described solidifying agent is organoalkoxysilane, after opening well 4 and probe filling orifice 5, after plasma treatment, with the permanent bonding of slide.
3, functionalization chip: the probe that target nucleic acid is corresponding, positive probe and negative probe are coated in amplification pond corresponding to chip 1 by Freeze Drying Technique respectively, complete the functionalization chip, and this chip can be 4
oC left and right prolonged preservation.
Embodiment
We utilize the multiple microfluid isothermal amplification technology of this array platform, have developed the multiple microfluid constant-temperature amplification of the array chip of human papilloma HPV virus 6/11,16,18 somatotypes of a Five-channel.In the micro-fluidic constant-temperature amplification chip of this five conduits array, we are 1,2, coated upper specific probe (Probe 6+11 for four kinds of important HPV goal gene in 3 amplification ponds, Probe 16 and Probe 18), in coated positive internal reference nucleic acid probe in amplification pond 4, amplification pond 5 as negative internal reference.We pass into 2 μ L samples, then splash into ~ 30 μ L LAMP reaction solutions, and the capping hole is 63
oUnder C, isothermal reaction 1 h(LAMP reaction solution composition sees Table 1).The macroscopic green that has occurred the LAMP signal in being coated with the amplification pond of target compound probe.We verify chip with the HPV sample respectively.Result is as follows: for sample HPV6/11, HPV 16 and HPV18 green amplified signal occurred in correspondent probe coated amplification pond, and negative internal reference contrasts the appearance that there is no amplified signal in the pond, result meets expection, and the crossed contamination between appearance amplification pond, and detected result is reliable.
Table 1 LAMP reaction solution composition
| Buffer MgSO4 Betaine dNTPs SYBR green Bst enzyme |
| 1× 8 mM 0.8M 1.0 mM 10 uM 8U |
The basic operational steps of chip is as follows:
1, sample injects: sampling, extract DNA, and with liquid-transfering gun, tested nucleic acid samples (2 μ L) is added to the chip addition pool, then splash into the approximately LAMP amplification liquid of 30 μ L.Under pulling force capillaceous, sample and amplification liquid distribute equably rapidly and enter the augmentation detection pond.
2, constant-temperature amplification: with the chip involution, carry out approximately 1 h of isothermal reaction in water-bath with uncured polydimethylsiloxane.
3, result judgement: can be directly also whether be transformed into the green detected result of judging according to reaction in the amplification pond, also can be under ultraviolet lamp come result of determination according to whether producing green fluorescence.
The present invention realizes effective differentiation of multiple LAMP signal by amplification pool space array sequence on chip, visual result, and successful need to by other expensive instruments, not be very beneficial for the LAMP method in the promotion and application of basic unit.By little processing chip manufacturing method, can realize as required any spatial multiplex array arrangement in amplification pond on chip.Has unlimited extensibility on this chip structure pattern theory, can completing according to the actual requirements arbitrarily, multiple LAMP effectively increases, realize the effectively amplification fast of many target spots, thereby satisfy effective quick diagnosis of clinical any multiple pathogens or heredopathia.Be no more than 1 h from being loaded onto the result judgement, and operation is very simple and convenient, the non-specialised staff just can successfully operate by simple demonstration.
＜110〉the Hangzhou bio tech ltd of jumping over
＜120〉be used for the multiple microfluid constant-temperature amplification of the array chip that high-risk HPV detects
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Claims (3)
1. one kind is used for the multiple microfluid constant-temperature amplification of the array chip that HPV detects, it is characterized in that, it comprises capillary channel (3), well (4), some amplifications ponds (1) and some probe filling orifices (5), described well (4) communicates with capillary channel (3), one end in all amplification ponds (1) all is communicated with capillary channel (3) by connecting tube (2), and each is communicated with the other end with a probe filling orifice (5); Wherein, be equipped with positive probe in an amplification pond (1), this positive probe has the DNA sequence dna shown in SEQ ID NO.25-SEQ ID NO.28, be equipped with negative probe in another pond of increasing, described negative probe is water, and described positive probe and negative probe are in order to improve the confidence level of detected result; Be coated with respectively upper specific probe for the target nucleic acid fragment in all the other ponds 1 of increasing; Described specific probe for the target nucleic acid fragment is for HPV-6,11 the probe with the DNA sequence dna shown in SEQ ID NO.1-SEQ ID NO.12, for the probe with the DNA sequence dna shown in SEQ ID NO.13-SEQ ID NO.18 of HPV-16 or for the probe with the DNA sequence dna shown in SEQ ID NO.19-SEQ ID NO.24 of HPV-18.
2. be used for according to claim 1 the multiple microfluid constant-temperature amplification of the array chip that HPV detects, it is characterized in that, described connecting tube (2) is quadrangular frustum pyramid shaped, the end that cross section is large is connected with amplification pond (1), the end that cross section is little is connected with capillary channel (3), make microfluid steadily flow into uniformly from capillary channel (3) pond (1) of increasing, and do not produce bubble.
3. be used for according to claim 1 the multiple microfluid constant-temperature amplification of the array chip that HPV detects, it is characterized in that, described amplification pool space size is according to its volume demand, and its length can change arbitrarily with demand, the general ￡ 1mm of the bulk of width and the degree of depth; The general ￡ 0.3mm of the width of capillary channel and the degree of depth can.
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Cited By (6)
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| CN103667011A (en) * | 2013-10-23 | 2014-03-26 | 国家纳米科学中心 | Micro-fluidic chip for loop-mediated isothermal amplification, preparation method and application of micro-fluidic chip |
| CN104593255A (en) * | 2015-02-06 | 2015-05-06 | 大连医科大学附属第二医院 | Microfluidic chip for instantly detecting EGFR (epidermal growth factor receptor) mutation |
| CN104805218A (en) * | 2015-04-02 | 2015-07-29 | 赣南医学院第一附属医院 | LAMP-and-molecular-beacon-based reaction system and method for detecting HPV16 and HPV18 |
| CN104862224A (en) * | 2015-05-18 | 2015-08-26 | 中国科学院深圳先进技术研究院 | Sliding digital PCR (polymerase chain reaction) chip with embedded guide rails and digital PCR method |
| CN106854674A (en) * | 2015-12-08 | 2017-06-16 | 上海交通大学 | A kind of nucleic acid high flux method for quick based on capillary microarray |
| CN111808744A (en) * | 2020-08-03 | 2020-10-23 | 齐鲁工业大学 | Portable full-automatic nucleic acid constant temperature amplification detector |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN104862224A (en) * | 2015-05-18 | 2015-08-26 | 中国科学院深圳先进技术研究院 | Sliding digital PCR (polymerase chain reaction) chip with embedded guide rails and digital PCR method |
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| CN111808744A (en) * | 2020-08-03 | 2020-10-23 | 齐鲁工业大学 | Portable full-automatic nucleic acid constant temperature amplification detector |
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