WO2016070761A1 - 结合压电传感与环介导恒温扩增的核酸检测板及检测系统 - Google Patents

结合压电传感与环介导恒温扩增的核酸检测板及检测系统 Download PDF

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WO2016070761A1
WO2016070761A1 PCT/CN2015/093556 CN2015093556W WO2016070761A1 WO 2016070761 A1 WO2016070761 A1 WO 2016070761A1 CN 2015093556 W CN2015093556 W CN 2015093556W WO 2016070761 A1 WO2016070761 A1 WO 2016070761A1
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nucleic acid
piezoelectric sensor
valve
detection system
acid detection
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PCT/CN2015/093556
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English (en)
French (fr)
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周连群
李传宇
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中国科学院苏州生物医学工程技术研究所
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Priority to US15/505,026 priority Critical patent/US10392591B2/en
Priority to JP2017511349A priority patent/JP2017529072A/ja
Publication of WO2016070761A1 publication Critical patent/WO2016070761A1/zh

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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C12M1/3407—Measure of electrical or magnetical factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/22—Details, e.g. general constructional or apparatus details
    • G01N29/222—Constructional or flow details for analysing fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/22—Details, e.g. general constructional or apparatus details
    • G01N29/24—Probes
    • G01N29/2437—Piezoelectric probes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0622—Valves, specific forms thereof distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0255—(Bio)chemical reactions, e.g. on biosensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/04—Wave modes and trajectories
    • G01N2291/042—Wave modes
    • G01N2291/0427—Flexural waves, plate waves, e.g. Lamb waves, tuning fork, cantilever

Definitions

  • the invention relates to a nucleic acid detecting device, in particular to a nucleic acid detecting plate and a detecting system for detecting blood and combining piezoelectric sensing and ring-mediated constant temperature amplification.
  • the bacterial culture method is currently the most accurate blood bacteria detection method, and is widely used in blood service institutions in Europe, the United States, and Canada.
  • the Bact/ALERT system determines the concentration of bacteria by continuously measuring the concentration of CO 2 in the culture flask. It is necessary to store the product for 24 hours before the test, so that the bacteria reach a certain amount, and it takes a certain time for the culture to reach a positive result, usually 72 hours or more.
  • Pall eBDS detects bacterial contamination by continuously measuring the consumption of O 2 in the sample bag.
  • the bacteria also need to be cultured for 24 hours. Therefore, the bacterial culture method takes a long time, the system configuration and maintenance cost are very high, and the method is prone to false negative results due to the hysteresis of bacterial growth.
  • the rapid detection method avoids the long-term defects of the bacterial culture method.
  • the Scansystem uses the monoclonal antibody to aggregate the platelets after filtration, and uses a penetrating agent and a fluorescent label for the bacterial DNA remaining in the sample, and the laser is scanned to determine the contamination and the positive report time is detected.
  • the shortening is 90min, but its sensitivity is only 10 3 CFU/mL.
  • the system needs to distinguish the fluorescent signal, which is more demanding for operators and difficult to promote.
  • the object of the present invention is to provide a nucleic acid detection board and a detection system capable of organically combining the advantages of piezoelectric sensing and ring-mediated constant temperature amplification (hereinafter referred to as constant temperature amplification). Improve the sensitivity and speed of nucleic acid detection, and reduce the operational difficulty and detection cost of nucleic acid detection.
  • nucleic acid is very easy to be contaminated. It is best to be arranged in a closed environment for testing, but this results in a long detection time, high cost, cumbersome operation steps, and high operator requirements. It is difficult to obtain a generalized application. Therefore, another object of the present invention is to provide a completely new hardware structure for nucleic acid detection, which enables one-stop rapid detection of nucleic acid detection in a fully enclosed environment without pauses in the middle, nucleic acid Nor is it in contact with the outside world.
  • a nucleic acid detecting plate comprising a piezoelectric sensor and at least one pipe flowing through a surface of the piezoelectric sensor, wherein the nucleic acid to be detected is introduced into the pipe in the form of a liquid and flows through the surface of the piezoelectric sensor;
  • Two valves are disposed at intervals between the pipeline and the upstream end of the piezoelectric sensor, and the nucleic acid to be tested is blocked in a conduit between the two valves for loop-mediated constant temperature amplification before flowing through the piezoelectric sensor, so that The amplified nucleic acid to be tested can be sensed by a piezoelectric sensor.
  • the nucleic acid detecting plate wherein the two valves are respectively a first valve close to the piezoelectric sensor and a second valve remote from the piezoelectric sensor, wherein an upstream end of the second valve
  • There is also a three-way valve and one of the three-way valves is connected with a sample cell for storing the nucleic acid to be tested, the auxiliary agent required for nucleic acid amplification, and the nucleic acid to be tested after being amplified.
  • the auxiliary agent sensed by the electrical sensor; the other port is connected to the outside of the nucleic acid detecting plate for introducing a washing liquid or a buffer into the pipe.
  • the nucleic acid detecting plate wherein the pipe is connected with a waste liquid tank with respect to a downstream end of the piezoelectric sensor, the waste liquid pool for recovering the nucleic acid to be detected sensed by the piezoelectric sensor to avoid Pollution.
  • a nucleic acid detection system comprising the nucleic acid detection plate as described above, and
  • An incubator capable of accommodating the nucleic acid detection plate
  • a signal processor capable of data connection with the piezoelectric sensor.
  • the nucleic acid detection system wherein the nucleic acid detection system further comprises a syringe pump for driving the liquid to flow in the conduit.
  • the nucleic acid detection system wherein the first valve, the second valve, and the three-way valve are electrically controlled valves;
  • An elevating table is disposed above the nucleic acid detecting plate, and a position of the first valve, the second valve and the three-way valve opposite to the first valve, the second valve and the three-way valve are respectively disposed on the lifting platform to selectively trigger The opening and closing of the electronically controlled valve.
  • the nucleic acid detecting system wherein a position corresponding to the piezoelectric sensor on the lifting platform is provided with a second telescopic probe to selectively control the piezoelectric sensor and the signal processor Data Connections.
  • the nucleic acid detection system is characterized in that an inlet is provided on the incubator directly opposite the sample cell.
  • the nucleic acid detection system wherein the inlet is filled with a seal.
  • the nucleic acid detecting system wherein the surface of the incubator is further provided with a threading hole for the through-tube wiring, the threading hole being filled with a sealing member.
  • the closed structure can avoid the problems of aerosol pollution that may be involved in nucleic acid detection, and is easy to realize mass production.
  • the detection system is suitable for solving the long-term detection time caused by existing blood pathogens, especially platelet bacteria pollution. Low volume, high cost, and cumbersome steps.
  • Figure 1 is a schematic view showing the structure of a nucleic acid detecting plate.
  • FIG. 2 is a schematic view showing the structure of a nucleic acid detection system.
  • FIG. 3 is a schematic view showing the structure of a lifting platform in a nucleic acid detecting system.
  • FIG. 4 is a schematic view showing the structure of an incubator in a nucleic acid detection system.
  • the present invention provides a nucleic acid detecting plate 1 of an embodiment comprising a piezoelectric sensor 2 and at least one pipe 3 flowing through the surface of the piezoelectric sensor.
  • a nucleic acid detecting plate 1 of an embodiment comprising a piezoelectric sensor 2 and at least one pipe 3 flowing through the surface of the piezoelectric sensor.
  • the specific form adopted can be unlimited, and it suffices that the nucleic acid to be tested can be brought into contact with the sensing surface of the piezoelectric sensor 2.
  • the piezoelectric sensor 2 is preferably a piezoelectric acoustic wave sensor, and more preferably a Lamb wave sensor.
  • Two valves are disposed at intervals between the pipe 3 and the upstream end of the piezoelectric sensor 2, and the nucleic acid to be tested is blocked in a pipe between the two valves for constant temperature amplification before flowing through the piezoelectric sensor 2, so that the expansion is performed.
  • the increased nucleic acid to be tested can be sensed by the piezoelectric sensor 2.
  • the purpose of isothermal amplification is to increase the size of the nucleic acid molecule so that it can be more easily sensed by the piezoelectric sensor 2.
  • the volume of the pipe can be calculated, so that the concentration of the nucleic acid to be measured can be easily converted.
  • the two valves are respectively the first valve 4 close to the piezoelectric sensor 2 and the second valve 5 remote from the piezoelectric sensor 2, wherein the upstream end of the second valve 5 is further provided with a three-way valve 6
  • a sample cell 7 is connected to one of the three-way valves 6, and the sample cell 7 is used for storing the nucleic acid to be tested, the auxiliary agent required for nucleic acid amplification, and the nucleic acid to be tested after the amplification is sensed by the piezoelectric sensor.
  • the auxiliary agent for assisting the amplified nucleic acid to be detected by the piezoelectric sensor is usually an antibody molecule for modifying the surface of the piezoelectric sensor; the surface of the piezoelectric sensor is modified to adsorb the nucleic acid molecule to be tested, of course Which specific modification molecule is used depends on the specific species of the nucleic acid to be tested.
  • Nucleic acid amplification The auxiliaries required and the nucleic acid to be tested after the amplification are coexisted by the sensitizer sensed by the piezoelectric sensor, and the two are not reacted before the nucleic acid to be tested is stored in the sample cell before the nucleic acid to be tested is injected.
  • the three-way valve 6 After entering the sample cell, the three are started to be driven into the pipeline between the first valve 4 and the second valve 5 to start amplification, and since the amplification speed is not fast, the transient coexistence of the three is amplified. No effect.
  • the other port of the three-way valve 6 is connected to the outside of the nucleic acid detecting plate 1 for introducing a washing liquid or a buffer into the pipe.
  • the function of the three-way valve 6 is to separately control the inflow of the liquid in the sample cell 7 with the buffer or the cleaning liquid to improve the automation of the detection and the detection efficiency.
  • the pipe 3 is connected with the waste liquid tank 8 with respect to the downstream end of the piezoelectric sensor 2, and the waste liquid pool 8 is for recovering the nucleic acid to be detected sensed by the piezoelectric sensor 2 to avoid contamination.
  • the present invention provides a nucleic acid detection system of an embodiment comprising the nucleic acid detection plate 1 as described above, and
  • An incubator 9 capable of accommodating the nucleic acid detecting plate 1;
  • a signal processor 10 capable of data connection with the piezoelectric sensor 2.
  • the nucleic acid detection system further includes a syringe pump 11 for driving the liquid to flow within the conduit.
  • the syringe pump 11 can be connected to the top of the waste liquid tank 8.
  • the first valve 4, the second valve 5, and the three-way valve 6 are all electrically controlled valves;
  • An elevating table 12 is disposed above the nucleic acid detecting plate 1, and a retractable first probe 13 is respectively disposed at a position opposite to the first valve 4, the second valve 5, and the three-way valve 6 on the lifting table 12 to select Slightly trigger the opening and closing of the electronically controlled valve.
  • This control can be controlled by an integrated control chip.
  • the control mode of the probe can be set freely. The setting can be selected. When the probe is in contact with the probe, the valve is opened, and vice versa. Alternatively, when the probe is in contact with the probe, the valve is closed, and vice versa. .
  • the control of the lifting platform can also select chip electric control, mechanical semi-automatic control or pure manual control according to actual needs.
  • an inlet 15 is provided in the incubator 9 opposite the sample cell 7.
  • the function of the inlet 15 is to facilitate injection.
  • the inlet 15 is filled with a seal.
  • the specific form or type of the seal is not limited, and it is mainly used to ensure the constant temperature effect of the incubator to ensure that the efficiency of the amplification meets the expected effect.
  • the surface of the incubator 9 is further provided with a threading hole 16 for the pipe-through wiring, which is filled with a seal.
  • the specific form or type of seal is also not limited.
  • the workflow of the nucleic acid detection system is:
  • the syringe pump 11 draws the sample to be tested into the pipeline between the first valve 4 and the second valve 5, closes all the valves, and the nucleic acid to be tested starts to be heated at a constant temperature;
  • the first valve 4, the second valve 5 and the three-way valve are connected to the branch of the buffer to introduce a buffer; at this time, the nucleic acid to be tested passes through the piezoelectric sensor 2, and the modifying molecule in the auxiliary agent will Adsorbing the nucleic acid to be tested to the surface of the sensor, causing a change in mass, thereby changing the vibration frequency of the sensor; the signal processor 10 acquires the phase and amplitude changes of the signal of the piezoelectric sensor 2 and records and saves;

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Abstract

一种结合压电传感与环介导恒温扩增的核酸检测板（1）及检测系统，包括压电传感器（2）和至少一条流经该压电传感器（2）表面的管道（3），在管道（3）相对于压电传感器（2）的上游端间隔设置两个阀门（4、5），待测核酸在流经压电传感器（2）之前被封锁在两个阀门（4、5）之间的管道（3）内进行恒温扩增；核酸检测系统包括上述核酸检测板（1）、能够容置该核酸检测板（1）的恒温箱（9）以及能够与压电传感器（2）数据连接的信号处理器（10）。由于采用了将恒温扩增和压电传感结合后进行联动检测，因此简化了装置结构、提高了检测效率；全程密闭的结构能够避开核酸检测中可能涉及的气溶胶污染等问题，并易于实现批量化生产，检测系统适用于解决现有血液病原引起的、尤其是血小板细菌污染引起的检测时间长、通量低、成本高、步骤繁琐等问题。

Description

结合压电传感与环介导恒温扩增的核酸检测板及检测系统 技术领域
本发明涉及一种核酸检测装置，特别涉及一种用于对血液进行检测且结合压电传感与环介导恒温扩增的核酸检测板及检测系统。
背景技术
输血相关传染病的预防和控制已经成为全社会关注的焦点，新技术的引进是进一步提高血液安全性的重要一环。在输血领域中，由于某些病原可能存在于捐献的血液和血液成份中，能通过输血传播引起受血者产生严重的临床后果，输血不可避免地存在某种程度的风险。并且相关的病原范围非常广泛，包括病毒、细菌、寄生虫和某种蛋白。其中经输血传播细菌的风险还很高，比如血小板制品细菌污染的危险度比病毒污染高出250倍。欧洲一些国家自1990年开始对所有血小板制品进行细菌检测，2004年，美国将血小板制品细菌检测列为必检项目。
细菌培养法是目前公认的最准确的血液细菌检测方法，在欧洲、美国、加拿大等国的血液服务机构被广泛使用。如Bact/ALERT系统通过持续检测培养瓶中CO2的浓度来判定细菌的浓度，检测前需将产品保存24h，让细菌达到一定数量，同时培养达到阳性结果也需要一定的时间，通常72h以上。Pall eBDS则是通过连续测量留样袋中O2的消耗量来检测细菌污染情况，细菌同样需要培养24h。所以，细菌培养法耗时长，对应系统配置及维护成本非常高，同时由于细菌生长具有迟滞性，该方法容易出现假阴性结果。
快速检测法避免了细菌培养法耗时长的缺陷，例如Scansystem利用单抗聚集血小板滤过后，对留在样品中的细菌DNA使用通透剂和荧光标记，经激光扫描确定污染情况，检测阳性报告时间缩短为90min，但是其灵敏度只有103CFU/mL，同时系统需要区分荧光信号，对操作人员要求较高，难以推广使用。目前，需开发一款灵敏度高、低成本、高通量的血小板细菌检测设备，来满足血液筛查和临床诊断对于输血安全的需要，降低血液细菌污染率，保证输血安全。
在核酸扩增方面，目前大多采用的是传统PCR法，但这种方法的缺点在于特异性低、灵敏度低、检测速度慢，并且该技术操作复杂，对仪器设备要求高，难以满足基层快速诊断的需求。
发明内容
针对现有技术中存在的不足之处，本发明的目的在于提供一种核酸检测板及检测系统，其能够有机结合压电传感与环介导恒温扩增(下文简称恒温扩增)的优势，提高对核酸检测的灵敏度和速度，降低核酸检测的操作难度和检测成本。
然而，核酸十分容易被污染，它最好能够全程都被安排在密闭环境下进行检测，但这就导致了它的检测时间长，成本高，且操作步骤繁琐，对操作者的要求也高，难以得到推广应用，因此，本发明的另一目的是提供一种全新的用于核酸检测的硬件结构，其能够使得将核酸检测在全密闭环境下实现一站式快速检测，中间无停顿，核酸也不与外界接触。
为实现上述目的，本发明通过以下技术方案实现：
一种核酸检测板，其包括压电传感器和至少一条流经该压电传感器表面的管道，待测核酸以液体的形式被引入至该管道内并流经所述压电传感器表面；
在所述管道相对于压电传感器的上游端间隔设置有两个阀门，待测核酸在流经压电传感器之前被封锁在两个阀门之间的管道内进行环介导恒温扩增，以使得经扩增后的待测核酸能够被压电传感器所感测。
优选的是，所述的核酸检测板，其中，所述两个阀门分别为靠近所述压电传感器的第一阀门和远离所述压电传感器的第二阀门，其中，第二阀门的上游端还设有一个三通阀，三通阀中的一个支口连接有一个样品池，该样品池用于存放待测核酸、核酸扩增所需助剂和帮助扩增后的待测核酸被压电传感器所感测的助剂；另一支口连接至所述核酸检测板的外部，用于向管道内引入清洗液或缓冲液。
优选的是，所述的核酸检测板，其中，所述管道相对于压电传感器的下游端连接有废液池，该废液池用于回收经压电传感器感测的待测核酸以避免 污染。
一种核酸检测系统，其包括有如上所述的核酸检测板，以及
能够容置该核酸检测板的恒温箱；以及
能够与所述压电传感器数据连接的信号处理器。
优选的是，所述的核酸检测系统，其中，所述核酸检测系统还包括有注射泵，其用于驱动液体在管道内流动。
优选的是，所述的核酸检测系统，其中，所述第一阀门、第二阀门和三通阀均为电控阀门；并且
在所述核酸检测板的上方设置一个升降台，在该升降台上与所述第一阀门、第二阀门和三通阀相对的位置分别设置有可伸缩的第一探针来选择性地触发电控阀门的开启和闭合。
优选的是，所述的核酸检测系统，其中，在所述升降台上与所述压电传感器相对的位置设置有可伸缩的第二探针来选择性地控制压电传感器与信号处理器的数据连接。
优选的是，所述的核酸检测系统，其中，在所述恒温箱上与所述样品池上方正对处开设有进样口。
优选的是，所述的核酸检测系统，其中，所述进样口填充有密封件。
优选的是，所述的核酸检测系统，其中，所述恒温箱的表面还设置有用于穿管布线的穿线孔，该穿线孔内填充有密封件。
本发明的有益效果是：
1)将恒温扩增和压电传感结合后进行联动检测，简化了装置结构，提高了检测效率，压电传感器的高特异性使得对核酸的检测更加灵敏，检测限达到了0.1pg/mL；
2)全程密闭的结构能够避开核酸检测中可能涉及的气溶胶污染等问题，并易于实现批量化生产，检测系统适用于解决现有血液病原引起尤其是血小板细菌污染引起的检测时间长、通量低、成本高、步骤繁琐等问题。
附图说明
图1为核酸检测板的结构示意图。
图2为核酸检测系统的结构示意图。
图3为核酸检测系统中升降台的结构示意图。
图4为核酸检测系统中恒温箱的结构示意图。
具体实施方式
下面结合附图对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。
参见图1～4，本案提供一实施例的核酸检测板1，其包括压电传感器2和至少一条流经该压电传感器表面的管道3，管道3可以有多条，但每条管道3之间是彼此独立的，以满足多个样品可以同时被检测；待测核酸以液体的形式被引入至该管道3内并流经压电传感器2表面；待测核酸流经压电传感器2表面所采用的具体形式可以不受限制，只需保证待测核酸能够与压电传感器2的感测面相接触即可。当管道3含有多条时，相应地，在压电传感器2的表面上，也应该设置有相同数量的彼此独立的感测面，以满足多通道检测需求。压电传感器2优选是压电声波传感器，进一步优选是Lamb波传感器。
在管道3相对于压电传感器2的上游端间隔设置有两个阀门，待测核酸在流经压电传感器2之前被封锁在两个阀门之间的管道内进行恒温扩增，以使得经扩增后的待测核酸能够被压电传感器2所感测。恒温扩增的目的是增加核酸分子的大小，以使得其可以更加容易被压电传感器2所吸附感测。两个阀门之间的距离只要是固定的，那么就可以计算出这段管道的体积，从而可以方便对待测核酸的浓度进行换算。
在上述实施例中，两个阀门分别为靠近压电传感器2的第一阀门4和远离压电传感器2的第二阀门5，其中，第二阀门5的上游端还设有一个三通阀6，三通阀6中的一个支口连接有一个样品池7，该样品池7用于存放待测核酸、核酸扩增所需助剂和帮助扩增后的待测核酸被压电传感器所感测的助剂；帮助扩增后的待测核酸被压电传感器所感测的助剂通常是一些对压电传感器表面进行修饰的抗体分子；压电传感器表面经修饰后可以吸附待测核酸分子，当然，具体采用何种修饰分子取决于待测核酸的具体种类。核酸扩增 所需助剂和帮助扩增后的待测核酸被压电传感器所感测的助剂两者共存时不会发生反应，它们两者先于待测核酸被存放在样品池内，当待测核酸注入进样品池后，三者即开始被驱动至第一阀门4和第二阀门5之间的管道内开始扩增，由于扩增速度不会很快，因此，三者短暂的共存对扩增结果没有影响。三通阀6的另一支口连接至核酸检测板1的外部，用于向管道内引入清洗液或缓冲液。三通阀6的作用是分别控制样品池7中的液体与缓冲液或清洗液的流入，以提高检测的自动化程度和检测效率。
在上述实施例中，管道3相对于压电传感器2的下游端连接有废液池8，该废液池8用于回收经压电传感器2感测的待测核酸以避免污染。
本案提供一实施例的核酸检测系统，其包括有如上所述的核酸检测板1，以及
能够容置该核酸检测板1的恒温箱9；以及
能够与压电传感器2数据连接的信号处理器10。
在该实施例中，核酸检测系统还包括有注射泵11，其用于驱动液体在管道内流动。注射泵11可以连接到废液池8的顶部。
在该实施例中，第一阀门4、第二阀门5和三通阀6均为电控阀门；并且
在核酸检测板1的上方设置一个升降台12，在该升降台12上与第一阀门4、第二阀门5和三通阀6相对的位置分别设置有可伸缩的第一探针13来选择性地触发电控阀门的开启和闭合。这种控制可通过集成的控制芯片来控制完成。探针的控制方式可以自由设定，可选择设定当探针下探与探头接触时，阀门打开，反之关闭；亦可选择设定当探针下探与探头接触时，阀门关闭，反之开启。此外，对升降台的控制也可根据实际需要选择芯片电动控制、机械半自动控制或纯手动控制。
在该实施例中，在升降台12上与压电传感器2相对的位置设置有可伸缩的第二探针14来选择性地控制压电传感器2与信号处理器10的数据连接。探针触碰到压电传感器的两端电极后，由于压电晶体存在逆压电效应，从而使压电传感器2薄膜处发生振动，再通过正压电效应使机械能转化为电能，产生输出电信号。该传感器根据传感器表面质量的改变引起振动频率的改变， 从而计算出待测液中待测核酸的含量。
在该实施例中，在恒温箱9上与样品池7上方正对处开设有进样口15。进样口15的作用是便于进样。
在该实施例中，进样口15填充有密封件。密封件的具体形式或种类不受限定，它主要用于保证恒温箱的恒温效果，以保证扩增的效率符合预期效果。
在该实施例中，恒温箱9的表面还设置有用于穿管布线的穿线孔16，该穿线孔16内填充有密封件。密封件的具体形式或种类也不受限定。
核酸检测系统的工作流程为：
1)打开恒温箱9的侧板，将核酸检测板1放入恒温箱9内，关闭侧板，开启恒温箱9进行升温，升温至扩增所需的温度；
2)阀门全部打开，关闭三通阀连接样品池7的那条支口，注射泵11驱动缓冲液引入到管道3内进行清洗；
3)关闭三通阀连接缓冲液的支口，打开连接样品池7的支口，第一阀门4和第二阀门5保持导通状态；从进样口15向样品池7内注入待测核酸液体；(助剂可以事先注入样品池7内)
4)注射泵11将待测样抽至第一阀门4和第二阀门5之间的管道内，关闭所有阀门，待测核酸开始恒温扩增；
5)扩增完成后，打开第一阀门4、第二阀门5和三通阀连接缓冲液的支口，引入缓冲液；此时待测核酸经过压电传感器2，助剂中的修饰分子会将待测核酸吸附至传感器表面，引起质量变化，从而改变传感器的振动频率；信号处理器10获取压电传感器2的信号相位和幅度变化并记录保存；
6)所有样品被抽至废液池8中，缓冲液清洗传感器表面；完成测试，丢弃核酸检测板1，换取新的核酸检测板1，开始下一轮的检测。附图中为10通道检测量，即单次单个检测板可以同时检测10个样品。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出与描述的图例。

Claims (10)

  1. 一种核酸检测板，其包括压电传感器和至少一条流经该压电传感器表面的管道，待测核酸以液体的形式被引入至该管道内并流经所述压电传感器表面；
    在所述管道相对于压电传感器的上游端间隔设置有两个阀门，待测核酸在流经压电传感器之前被封锁在两个阀门之间的管道内进行环介导恒温扩增，以使得经扩增后的待测核酸能够被压电传感器所感测。
  2. 根据权利要求1所述的核酸检测板，其特征在于，所述两个阀门分别为靠近所述压电传感器的第一阀门和远离所述压电传感器的第二阀门，其中，第二阀门的上游端还设有一个三通阀，三通阀中的一个支口连接有一个样品池，该样品池用于存放待测核酸、核酸扩增所需助剂和帮助扩增后的待测核酸被压电传感器所感测的助剂；另一支口连接至所述核酸检测板的外部，用于向管道内引入清洗液或缓冲液。
  3. 根据权利要求2所述的核酸检测板，其特征在于，所述管道相对于压电传感器的下游端连接有废液池，该废液池用于回收经压电传感器感测的待测核酸以避免污染。
  4. 一种核酸检测系统，其包括有如权利要求3所述的核酸检测板，以及
    能够容置该核酸检测板的恒温箱；以及
    能够与所述压电传感器数据连接的信号处理器。
  5. 根据权利要求4所述的核酸检测系统，其特征在于，所述核酸检测系统还包括有注射泵，其用于驱动液体在管道内流动。
  6. 根据权利要求4所述的核酸检测系统，其特征在于，所述第一阀门、第二阀门和三通阀均为电控阀门；并且
    在所述核酸检测板的上方设置一个升降台，在该升降台上与所述第一阀门、第二阀门和三通阀相对的位置分别设置有可伸缩的第一探针来选择性地触发电控阀门的开启和闭合。
  7. 根据权利要求6所述的核酸检测系统，其特征在于，在所述升降台上与所述压电传感器相对的位置设置有可伸缩的第二探针来选择性地控制压电传感器与信号处理器的数据连接。
  8. 根据权利要求4所述的核酸检测系统，其特征在于，在所述恒温箱上与所述样品池上方正对处开设有进样口。
  9. 根据权利要求8所述的核酸检测系统，其特征在于，所述进样口填充有密封件。
  10. 根据权利要求4所述的核酸检测系统，其特征在于，所述恒温箱的表面还设置有用于穿管布线的穿线孔，该穿线孔内填充有密封件。
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