CN111909835A - Closed micro-fluidic nucleic acid detection card box - Google Patents
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Abstract
本发明公开了一种封闭式微流控核酸检测卡盒，包括卡盒本体、裂解管、扩增管、核酸提取腔、废液腔、混合腔、第一清洗液存储腔、第二清洗液存储腔、洗脱液存储腔、第一油腔、第二油腔、第一机械旋转阀、第二机械旋转阀、第三机械旋转阀、第一气源通道、第二气源通道、第三气源通道、第四气源通道，整体形成一个封闭空间。本发明提供的一种集成核酸提取、扩增、检测功能于一体的封闭式卡盒，集成了核酸提取和扩增检测两个步骤，在一个小的密闭的盒体内完成，可用于没有分子诊断实验室环境下的核酸提取和扩增检测，具有结构紧凑、易于操作、结构简单、易装配等优点，有效提高分子诊断类产品的环境适应度。
The invention discloses a closed microfluidic nucleic acid detection cartridge, comprising a cartridge body, a lysis tube, an amplification tube, a nucleic acid extraction cavity, a waste liquid cavity, a mixing cavity, a first cleaning liquid storage cavity, and a second cleaning liquid storage cavity chamber, eluent storage chamber, first oil chamber, second oil chamber, first mechanical rotary valve, second mechanical rotary valve, third mechanical rotary valve, first air source channel, second air source channel, third The air source channel and the fourth air source channel form a closed space as a whole. The present invention provides a closed cartridge integrating nucleic acid extraction, amplification and detection functions, which integrates two steps of nucleic acid extraction and amplification detection, and is completed in a small closed box, which can be used for molecular diagnosis without molecular diagnosis. Nucleic acid extraction and amplification detection in the laboratory environment has the advantages of compact structure, easy operation, simple structure, and easy assembly, which can effectively improve the environmental adaptability of molecular diagnostic products.
Description
技术领域technical field
本发明涉及一种封闭式微流控核酸检测卡盒，特别涉及一种集成核酸提取、扩增、检测功能于一体的一种封闭式微流控核酸检测卡盒。The invention relates to a closed microfluidic nucleic acid detection cartridge, in particular to a closed microfluidic nucleic acid detection cartridge integrating nucleic acid extraction, amplification and detection functions.
背景技术Background technique
随着分子生物学的发展，体外快速扩增目的DNA/RNA片段的聚合酶链式反应(Polymerase Chain Reaction，PCR)技术获得广泛应用，已成为病原体基因快速检测的主要方法。实时荧光PCR技术通过引入荧光染料或荧光标记的特异性探针，对PCR过程进行实时监控，实现了从定性判断到定量分析的飞跃，能对病原体基因进行高效、特异性检测，大大缩短了检出窗口期，有利于实现早诊断、早治疗，降低病死率。With the development of molecular biology, the polymerase chain reaction (PCR) technology for rapidly amplifying target DNA/RNA fragments in vitro has been widely used and has become the main method for rapid detection of pathogen genes. Real-time fluorescent PCR technology realizes a leap from qualitative judgment to quantitative analysis by introducing fluorescent dyes or fluorescently labeled specific probes to monitor the PCR process in real time. The window-out period is conducive to early diagnosis, early treatment, and reduced mortality.
核酸检测周期短，灵敏度特异性与培养法相当，但存在以下的问题：1)将包括临床样品裂解释放核酸、核酸吸附、洗涤杂质、洗脱核酸，配置PCR反应体系以及核酸扩增分开进行，不仅操作步骤繁杂，而且耗时长；2)多设备联用，需要专业实验室及人员，过多的人工操作还易造成样品的交叉污染，进而造成假阴性或假阳性，同时检验人员被感染的风险倍增，严重制约了这项技术的应用和推广。The nucleic acid detection cycle is short, and the sensitivity and specificity are comparable to those of the culture method, but there are the following problems: 1) The clinical sample lysis to release nucleic acid, nucleic acid adsorption, washing of impurities, elution of nucleic acid, configuration of PCR reaction system and nucleic acid amplification are carried out separately, Not only the operation steps are complicated, but also time-consuming; 2) The combined use of multiple equipment requires professional laboratories and personnel. Excessive manual operation can also easily lead to cross-contamination of samples, resulting in false negatives or false positives. At the same time, inspectors are infected. The risk is multiplied, which seriously restricts the application and promotion of this technology.
发明内容SUMMARY OF THE INVENTION
本发明要解决的技术问题为：本发明通过微流控卡盒芯片的模式，解决传统核酸检测操作步骤繁杂、耗时长、需要人工干扰等技术问题。The technical problem to be solved by the present invention is as follows: the present invention solves the technical problems of traditional nucleic acid detection operation steps such as complicated operation steps, long time-consuming, manual interference and the like through the mode of microfluidic cartridge chip.
本发明解决上述技术问题采用的技术方案为，一种封闭式微流控核酸检测卡盒，包括卡盒本体、裂解管、扩增管、核酸提取腔、废液腔、混合腔、第一清洗液存储腔、第二清洗液存储腔、洗脱液存储腔、第一油腔、第二油腔、第一机械旋转阀、第二机械旋转阀、第三机械旋转阀、第一气源通道、第二气源通道、第三气源通道、第四气源通道、第一油封通道、第二油封通道、第三油封通道、第四油封通道、第一裂解液转移通道、第二裂解液转移通道、废液转移通道、混合通道、第一Z型试剂注入通道、第二Z型试剂注入通道、第三Z型试剂注入通道、第一洗脱液转移通道、第二洗脱液转移通道、第一气源端口、第二气源端口、第三气源端口，整体形成一个封闭空间；The technical solution adopted by the present invention to solve the above technical problems is: a closed microfluidic nucleic acid detection cartridge, comprising a cartridge body, a lysis tube, an amplification tube, a nucleic acid extraction chamber, a waste liquid chamber, a mixing chamber, and a first cleaning solution storage cavity, second cleaning liquid storage cavity, eluent storage cavity, first oil cavity, second oil cavity, first mechanical rotary valve, second mechanical rotary valve, third mechanical rotary valve, first air source channel, The second gas source channel, the third gas source channel, the fourth gas source channel, the first oil seal channel, the second oil seal channel, the third oil seal channel, the fourth oil seal channel, the first cracking liquid transfer channel, the second cracking liquid transfer channel, waste liquid transfer channel, mixing channel, first Z-type reagent injection channel, second Z-type reagent injection channel, third Z-type reagent injection channel, first eluent transfer channel, second eluent transfer channel, The first air source port, the second air source port, and the third air source port form a closed space as a whole;
进一步地，第一Z型试剂注入通道连接核酸提取腔与第一清洗液存储腔，第一清洗液存储腔中的试剂可以在外接动力源的作用下顺着第一Z型试剂注入通道进入核酸提取腔；第二Z型试剂注入通道连接核酸提取腔与第二清洗液存储腔，第二清洗液存储腔中的试剂可以在外接动力源的作用下顺着第二Z型试剂注入通道进入核酸提取腔；第三Z型试剂注入通道连接核酸提取腔与洗脱液存储腔，洗脱液存储腔中的试剂可以在外接动力源的作用下顺着第三Z型试剂注入通道进入核酸提取腔；Further, the first Z-shaped reagent injection channel connects the nucleic acid extraction chamber and the first cleaning solution storage chamber, and the reagents in the first cleaning solution storage chamber can enter the nucleic acid along the first Z-shaped reagent injection channel under the action of an external power source. Extraction chamber; the second Z-shaped reagent injection channel connects the nucleic acid extraction chamber and the second cleaning solution storage chamber, and the reagent in the second cleaning solution storage chamber can enter the nucleic acid along the second Z-shaped reagent injection channel under the action of an external power source Extraction chamber; the third Z-shaped reagent injection channel connects the nucleic acid extraction chamber and the eluent storage chamber, and the reagent in the eluent storage chamber can enter the nucleic acid extraction chamber along the third Z-shaped reagent injection channel under the action of an external power source ;
进一步地，所述第一气源通道、第二气源通道、第一油封通道、第二油封通道、第一裂解液转移通道、第二裂解液转移通道的通断使用第一机械旋转阀进行切换；Further, the on-off of the first gas source channel, the second gas source channel, the first oil seal channel, the second oil seal channel, the first cracking liquid transfer channel, and the second cracking liquid transfer channel is performed using a first mechanical rotary valve. switch;
进一步地，所述第一洗脱液转移通道、第二洗脱液转移通道、第三油封通道、第四油封通道、第三气源通道、第四气源通道的通断使用第二机械旋转阀进行切换；Further, the on-off of the first eluent transfer channel, the second eluent transfer channel, the third oil seal channel, the fourth oil seal channel, the third air source channel, and the fourth air source channel uses a second mechanical rotation valve to switch;
进一步地，所述第三机械旋转阀上设计有大气端口、第三气源端口。第三机械旋转阀可以围绕第大气端口进行旋转运动。大气端口始终与核酸提取腔所属核酸提取腔端口相互对接。第三机械旋转阀旋转不同角度时，第三气源端口将分别与第一清洗液存储腔所属第一清洗液存储腔端口、第二清洗液存储腔所属第二清洗液存储腔端口、洗脱液存储腔所属洗脱液存储腔端口、混合腔所属混合腔端口、废液腔所属废液腔端口相互对接。Further, an atmosphere port and a third air source port are designed on the third mechanical rotary valve. The third mechanical rotary valve may perform rotational movement around the first atmospheric port. The atmospheric port is always docked with the port of the nucleic acid extraction chamber to which the nucleic acid extraction chamber belongs. When the third mechanical rotary valve rotates at different angles, the third air source port will be respectively connected with the first cleaning liquid storage chamber port to which the first cleaning liquid storage chamber belongs, the second cleaning liquid storage chamber port to which the second cleaning liquid storage chamber belongs, and the elution The eluent storage chamber port to which the liquid storage chamber belongs, the mixing chamber port to which the mixing chamber belongs, and the waste liquid chamber port to which the waste liquid chamber belongs are butted with each other.
进一步地，核酸提取和检测时，转动第一机械旋转阀、第二机械旋转阀和第三机械旋转阀到不同的位置，进而导通不同的试剂通道、气路通道、油路通道和各类腔体，然后外接动力源提供动力给卡盒本体上端的第一气源端口、第二气源端口或第三气源端口，进而控制卡盒内的试剂流动，即将裂解管中的被裂解的样本转移进入核酸提取腔，随后在核酸提取腔内完成磁珠吸附核酸、磁珠清洗、废液转移至废液腔、核酸洗脱、转移核酸至扩增管核酸提取和检测过程，最后在扩增管中完成检测。Further, during nucleic acid extraction and detection, turn the first mechanical rotary valve, the second mechanical rotary valve and the third mechanical rotary valve to different positions, thereby conducting different reagent channels, gas channel channels, oil channel channels and various The cavity, and then the external power source provides power to the first gas source port, the second gas source port or the third gas source port on the upper end of the cartridge body, and then controls the flow of reagents in the cartridge, that is, the cracked gas in the cracking tube. The sample is transferred into the nucleic acid extraction chamber, and then the magnetic bead adsorption of nucleic acid, magnetic bead cleaning, waste liquid transfer to the waste liquid chamber, nucleic acid elution, nucleic acid transfer to the amplification tube nucleic acid extraction and detection process are completed in the nucleic acid extraction chamber, and finally the amplification process is performed. The test is completed in the expansion pipe.
所述卡盒本体上设置有第一油腔、第二油腔，油腔内部预埋有密封油，密封油可选用各类对下游扩增反应无抑制的、密度小于水的矿物油或者相似的物质；第一油腔顶部设计有第一油腔端口，与大气相通；第二油腔顶部设计有第二油腔端口，与大气相通。The cartridge body is provided with a first oil cavity and a second oil cavity, and a sealing oil is pre-buried in the oil cavity. The sealing oil can be selected from various types of mineral oil that does not inhibit the downstream amplification reaction and whose density is lower than that of water or similar. The top of the first oil chamber is designed with a first oil chamber port, which communicates with the atmosphere; the top of the second oil chamber is designed with a second oil chamber port, which communicates with the atmosphere.
所述卡盒本体上设置有核酸提取腔、废液腔、混合腔、第一清洗液存储腔、第二清洗液存储腔、洗脱液存储腔；The cartridge body is provided with a nucleic acid extraction cavity, a waste liquid cavity, a mixing cavity, a first cleaning liquid storage cavity, a second cleaning liquid storage cavity, and an eluent storage cavity;
进一步地，所述核酸提取腔顶部设计有核酸提取腔端口，与大气相通；Further, a nucleic acid extraction chamber port is designed on the top of the nucleic acid extraction chamber, which communicates with the atmosphere;
进一步地，所述废液腔顶部设计有废液腔端口，可以与第三机械旋转阀所属第二气源端口相通；Further, the top of the waste liquid cavity is designed with a waste liquid cavity port, which can communicate with the second gas source port to which the third mechanical rotary valve belongs;
进一步地，所述混合腔顶部设计有混合腔端口，可以与第三机械旋转阀所属第二气源端口相通；Further, the top of the mixing chamber is designed with a mixing chamber port, which can communicate with the second air source port to which the third mechanical rotary valve belongs;
进一步地，所述第一清洗液存储腔顶部设计有第一清洗液存储腔端口，可以与第三机械旋转阀所属第二气源端口相通；Further, the top of the first cleaning liquid storage chamber is designed with a first cleaning liquid storage chamber port, which can communicate with the second gas source port to which the third mechanical rotary valve belongs;
进一步地，所述第二清洗液存储腔顶部设计有第二清洗液存储腔端口，可以与第三机械旋转阀所属第二气源端口相通；Further, the top of the second cleaning liquid storage chamber is designed with a second cleaning liquid storage chamber port, which can communicate with the second gas source port to which the third mechanical rotary valve belongs;
进一步地，所述洗脱液存储腔顶部设计有洗脱液存储腔端口，可以与第三机械旋转阀所属第二气源端口相通。Further, the top of the eluent storage chamber is designed with an eluent storage chamber port, which can communicate with the second gas source port to which the third mechanical rotary valve belongs.
进一步地，所述核酸提取腔通过混合通道与混合腔相互连通；Further, the nucleic acid extraction chamber communicates with the mixing chamber through a mixing channel;
进一步地，所述核酸提取腔通过废液转移通道与废液腔相互连通；Further, the nucleic acid extraction cavity is communicated with the waste liquid cavity through a waste liquid transfer channel;
所述核酸提取腔通过第一Z型试剂注入通道与第一清洗液存储腔相互连通；The nucleic acid extraction chamber is communicated with the first cleaning liquid storage chamber through the first Z-shaped reagent injection channel;
进一步地，所述核酸提取腔通过第二Z型试剂注入通道与第二清洗液存储腔相互连通；Further, the nucleic acid extraction chamber communicates with the second cleaning solution storage chamber through the second Z-shaped reagent injection channel;
进一步地，所述核酸提取腔通过第三Z型试剂注入通道与洗脱液存储腔相互连通；Further, the nucleic acid extraction chamber communicates with the eluent storage chamber through a third Z-shaped reagent injection channel;
所述卡盒本体中部设置有两个机械旋转阀，即第一机械旋转阀、第二机械旋转阀。Two mechanical rotary valves are arranged in the middle of the cartridge body, namely a first mechanical rotary valve and a second mechanical rotary valve.
进一步地，所述第一机械旋转阀内部设置有第一转动杆安装处、第一阀内油封通道、第一阀内气源通道、第一阀内裂解液转移通道。Further, the first mechanical rotary valve is provided with a first rotating rod installation place, a first valve oil seal channel, a first valve gas source channel, and a first valve cracking liquid transfer channel.
进一步地，所述第二机械旋转阀内部设置有第二转动杆安装处、第二阀内油封通道、第二阀内气源通道、第一阀内洗脱液转移通道。Further, the second mechanical rotary valve is provided with a second rotating rod installation place, an oil seal passage in the second valve, an air source passage in the second valve, and an eluent transfer passage in the first valve.
所述第一机械旋转阀处于第一位置时，第一气源通道和第二气源通道、第一油封通道和第二油封通道、第一裂解液转移通道和第二裂解液转移通道处于关断状态；When the first mechanical rotary valve is in the first position, the first gas source channel and the second gas source channel, the first oil seal channel and the second oil seal channel, the first cracking liquid transfer channel and the second cracking liquid transfer channel are closed. off state;
进一步地，所述第一机械旋转阀处于第二位置时，第一油封通道和第二油封通道，第一气源通道和第二气源通道处于开通状态；第一裂解液转移通道和第二裂解液转移通道处于关断状态。此时，第一油腔端口、第一油腔、第一油封通道、第一阀内油封通道、第二油封通道、裂解管、第二气源通道、第一阀内气源通道、第一气源通道、第一气源端口相通。此时，外接动力源提供动力给予第一气源端口负压力，即可实现第一油腔中的密封油液转移进入裂解管的操作，密封裂解液，防止气溶胶污染。Further, when the first mechanical rotary valve is in the second position, the first oil seal channel and the second oil seal channel, the first gas source channel and the second gas source channel are in an open state; the first cracking liquid transfer channel and the second The lysate transfer channel is turned off. At this time, the first oil chamber port, the first oil chamber, the first oil seal channel, the first oil seal channel in the first valve, the second oil seal channel, the cracking tube, the second gas source channel, the first valve gas source channel, the first The air source channel and the first air source port are communicated. At this time, the external power source provides power to give negative pressure to the first gas source port, and the operation of transferring the sealed oil in the first oil chamber into the cracking tube can be realized, and the cracking liquid is sealed to prevent aerosol pollution.
进一步地，所述第一机械旋转阀处于第三位置时，第一气源通道和第二气源通道、第一裂解液转移通道和第二裂解液转移通道处于开通状态；第一油封通道和第二油封通道仍然处于关断状态。此时，第一气源端口、第一气源通道、第一阀内气源通道、第二气源通道、裂解管、第一裂解管转移通道、第一阀内裂解液通道、第二裂解管转移通道、核酸提取腔、核酸提取腔端口、第三机械旋转阀所属大气端口连通。此时，外接动力源提供动力给予第一气源端口正压力，即可实现裂解管中的裂解液转移进入核酸提取腔的操作。Further, when the first mechanical rotary valve is in the third position, the first gas source channel and the second gas source channel, the first cracking liquid transfer channel and the second cracking liquid transfer channel are in an open state; the first oil seal channel and The second oil seal passage is still closed. At this time, the first gas source port, the first gas source channel, the first gas source channel in the valve, the second gas source channel, the cracking tube, the first cracking tube transfer channel, the first cracking liquid channel in the valve, the second cracking tube The tube transfer channel, the nucleic acid extraction chamber, the port of the nucleic acid extraction chamber, and the atmospheric port to which the third mechanical rotary valve belongs are communicated. At this time, the external power source provides power to give positive pressure to the first gas source port, and the operation of transferring the lysate in the lysis tube into the nucleic acid extraction chamber can be realized.
进一步地，所述第二机械旋转阀处于第一位置时，第一洗脱液转移通道和第二洗脱液转移通道、第三油封通道和第四油封通道、第三气源通道和第四气源通道处于关断状态；Further, when the second mechanical rotary valve is in the first position, the first eluent transfer channel and the second eluent transfer channel, the third oil seal channel and the fourth oil seal channel, the third air source channel and the fourth The air source channel is closed;
进一步地，所述第二机械旋转阀处于第二位置时，第一洗脱液转移通道和第二洗脱液转移通道、第三气源通道和第四气源通道处于开通状态；第三油封通道和第四油封通道仍然处于关断状态。此时，第三气源端口、第三气源通道、第二阀内气源通道、第四气源通道、扩增管、第二洗脱液转移通道、第一阀内洗脱液转移通道、第一洗脱液转移通道、核酸提取腔、核酸提取腔端口、第三机械旋转阀所属大气端口连通。此时，外接动力源提供动力给予第三气源端口负压力，即可实现核酸提取腔的洗脱液转移进入扩增管的操作。Further, when the second mechanical rotary valve is in the second position, the first eluent transfer channel and the second eluent transfer channel, the third air source channel and the fourth air source channel are in an open state; the third oil seal The channel and the fourth oil seal channel are still closed. At this time, the third gas source port, the third gas source channel, the second gas source channel in the valve, the fourth gas source channel, the amplification tube, the second eluent transfer channel, and the first valve eluent transfer channel , the first eluent transfer channel, the nucleic acid extraction chamber, the port of the nucleic acid extraction chamber, and the atmospheric port to which the third mechanical rotary valve belongs are connected. At this time, the external power source provides power to give negative pressure to the third gas source port, so that the operation of transferring the eluate in the nucleic acid extraction chamber into the amplification tube can be realized.
进一步地，所述第二机械旋转阀处于第三位置时，第三油封通道和第四油封通道，第三气源通道和第四气源通道处于开通状态；第一洗脱液转移通道和第二洗脱液转移通道处于关断状态。此时，第三气源端口、第三气源通道、第二阀内气源通道、第四气源通道、扩增管、第四油封通道、第二阀内油封通道、第三油封通道、第二油腔、第二油腔端口相互连通。此时，外接动力源提供动力给予第三气源端口负压力，即可实现第二油腔的密封油转移进入扩增管的操作，密封扩增试剂，防止气溶胶污染。Further, when the second mechanical rotary valve is in the third position, the third oil seal channel and the fourth oil seal channel, the third air source channel and the fourth air source channel are in the open state; the first eluent transfer channel and the third The second eluent transfer channel is closed. At this time, the third air source port, the third air source channel, the second air source channel in the valve, the fourth air source channel, the amplification tube, the fourth oil seal channel, the second oil seal channel in the valve, the third oil seal channel, The second oil chamber and the ports of the second oil chamber communicate with each other. At this time, the external power source provides power to give negative pressure to the third gas source port, so that the operation of transferring the sealing oil of the second oil chamber into the amplification tube can be realized, and the amplification reagent can be sealed to prevent aerosol pollution.
所述卡盒本体顶部设置有一个机械旋转阀，即第三机械旋转阀。The top of the cartridge body is provided with a mechanical rotary valve, namely the third mechanical rotary valve.
进一步地，所述第三机械旋转阀内部设置有第三转动杆安装处、第二气源端口、大气端口。Further, the third mechanical rotary valve is provided with a third rotating rod installation place, a second air source port, and an atmosphere port.
进一步地，所述第三机械旋转阀以核酸提取腔所属核酸提取腔端口为中心进行旋转运动。Further, the third mechanical rotary valve rotates around the port of the nucleic acid extraction chamber to which the nucleic acid extraction chamber belongs.
进一步地，所述第三机械旋转阀处于第一位置时，第三机械旋转阀所属大气端口始终与核酸提取腔端口同心对准。此种状态下，第二气源端口与废液腔所属废液腔端口、混合腔所属废液腔端口、第一清洗液存储腔所属第一清洗液存储腔端口、第二清洗液存储腔所属第二清洗液存储腔端口、洗脱液存储腔所属洗脱液存储腔端口处于关断状态。Further, when the third mechanical rotary valve is in the first position, the atmospheric port to which the third mechanical rotary valve belongs is always concentrically aligned with the nucleic acid extraction chamber port. In this state, the second gas source port and the waste liquid chamber port to which the waste liquid chamber belongs, the waste liquid chamber port to which the mixing chamber belongs, the first cleaning liquid storage chamber port to which the first cleaning liquid storage chamber belongs, and the second cleaning liquid storage chamber to which the second cleaning liquid storage chamber belongs. The port of the second cleaning liquid storage chamber and the port of the eluent storage chamber to which the eluent storage chamber belongs are in a closed state.
进一步地，所述第三机械旋转阀处于第二位置时，第二气源端口与混合腔所属混合腔端口、混合腔、混合通道、核酸提取腔、核酸提取腔端口、大气端口处于连通状态；此时，第二气源端口与废液腔所属废液腔端口、第一清洗液存储腔所属第一清洗液存储腔端口、第二清洗液存储腔所属第二清洗液存储腔端口、洗脱液存储腔所属洗脱液存储腔端口处于关断状态。此时，外接动力源提供交替的正压力和负压力给予第二气源通道，即可实现核酸提取腔与混合腔中试剂的混合操作。Further, when the third mechanical rotary valve is in the second position, the second air source port and the mixing chamber port, the mixing chamber, the mixing channel, the nucleic acid extraction chamber, the nucleic acid extraction chamber port, and the atmosphere port to which the mixing chamber belongs are in a connected state; At this time, the second gas source port and the waste liquid chamber port to which the waste liquid chamber belongs, the first cleaning liquid storage chamber port to which the first cleaning liquid storage chamber belongs, the second cleaning liquid storage chamber port to which the second cleaning liquid storage chamber belongs, and the elution The port of the eluent storage chamber to which the liquid storage chamber belongs is off. At this time, the external power source provides alternating positive pressure and negative pressure to the second gas source channel, so that the mixing operation of the reagents in the nucleic acid extraction chamber and the mixing chamber can be realized.
进一步地，所述第三机械旋转阀处于第三位置时，第二气源端口与废液腔所属废液腔端口、废液腔、废液转移通道、核酸提取腔、核酸提取腔端口、大气端口处于连通状态；此时，第二气源端口与混合腔所属废液腔端口、第一清洗液存储腔所属第一清洗液存储腔端口、第二清洗液存储腔所属第二清洗液存储腔端口、洗脱液存储腔所属洗脱液存储腔端口处于关断状态。此时，外接动力源提供负压力给予第二气源通道，即可实现核酸提取腔中废液的转移操作。Further, when the third mechanical rotary valve is in the third position, the second gas source port and the waste liquid chamber belong to the waste liquid chamber port, the waste liquid chamber, the waste liquid transfer channel, the nucleic acid extraction chamber, the nucleic acid extraction chamber port, and the atmosphere. The ports are in a connected state; at this time, the second air source port and the waste liquid chamber port to which the mixing chamber belongs, the first cleaning liquid storage chamber port to which the first cleaning liquid storage chamber belongs, and the second cleaning liquid storage chamber to which the second cleaning liquid storage chamber belongs The port and the port of the eluent storage chamber to which the eluent storage chamber belongs are off. At this time, the external power source provides negative pressure to the second gas source channel, and the transfer operation of the waste liquid in the nucleic acid extraction chamber can be realized.
进一步地，所述第三机械旋转阀处于第四位置时，第二气源端口与第一清洗液存储腔所属第一清洗液存储腔端口、第一清洗液存储腔、第一Z型试剂注入通道、核酸提取腔、核酸提取腔端口、大气端口处于连通状态；此时，第二气源端口与废液腔所属废液腔端口、混合腔所属混合腔端口、第二清洗液存储腔所属第二清洗液存储腔端口、洗脱液存储腔所属洗脱液存储腔端口处于关断状态。此时，外接动力源提供正压力给予第二气源通道，即可实现第一清洗液存储腔内的第一清洗液注入核酸提取腔的操作。Further, when the third mechanical rotary valve is in the fourth position, the second air source port and the first cleaning fluid storage chamber port, the first cleaning fluid storage chamber, and the first Z-type reagent injection belong to the first cleaning fluid storage chamber. The channel, the nucleic acid extraction chamber, the nucleic acid extraction chamber port, and the air port are in a connected state; at this time, the second gas source port and the waste liquid chamber port to which the waste liquid chamber belongs, the mixing chamber port to which the mixing chamber belongs, and the second cleaning liquid storage chamber to which the second cleaning liquid storage chamber belongs. The port of the second cleaning solution storage chamber and the port of the eluent storage chamber to which the eluent storage chamber belongs are in a closed state. At this time, the external power source provides positive pressure to the second air source channel, so that the operation of injecting the first cleaning solution in the first cleaning solution storage chamber into the nucleic acid extraction chamber can be realized.
进一步地，所述第三机械旋转阀处于第五位置时，第二气源端口与第二清洗液存储腔所属第二清洗液存储腔端口、第二清洗液存储腔、第二Z型试剂注入通道、核酸提取腔、核酸提取腔端口、大气端口处于连通状态；此时，第二气源端口与废液腔所属废液腔端口、混合腔所属混合腔端口、第一清洗液存储腔所属第一清洗液存储腔端口、洗脱液存储腔所属洗脱液存储腔端口处于关断状态。此时，外接动力源提供正压力给予第二气源通道，即可实现第二清洗液存储腔内的第二清洗液注入核酸提取腔的操作。Further, when the third mechanical rotary valve is in the fifth position, the second air source port and the second cleaning fluid storage chamber belong to the second cleaning fluid storage chamber port, the second cleaning fluid storage chamber, and the second Z-type reagent injection. The channel, the nucleic acid extraction chamber, the nucleic acid extraction chamber port, and the air port are in a connected state; at this time, the second gas source port and the waste liquid chamber port to which the waste liquid chamber belongs, the mixing chamber port to which the mixing chamber belongs, and the first cleaning liquid storage chamber to which the first cleaning liquid storage chamber belongs. A port of the cleaning liquid storage chamber and the port of the eluent storage chamber to which the eluent storage chamber belongs are in a closed state. At this time, the external power source provides positive pressure to the second gas source channel, so that the operation of injecting the second cleaning solution in the second cleaning solution storage chamber into the nucleic acid extraction chamber can be realized.
进一步地，所述第三机械旋转阀处于第六位置时，第二气源端口与洗脱液存储腔所属洗脱液存储腔端口、洗脱液存储腔、第三Z型试剂注入通道、核酸提取腔、核酸提取腔端口、大气端口处于连通状态；此时，第二气源端口与废液腔所属废液腔端口、混合腔所属混合腔端口、第一清洗液存储腔所属第一清洗液存储腔端口、第二清洗液存储腔所属第二清洗液存储腔端口处于关断状态。此时，外接动力源提供正压力给予第二气源通道，即可实现洗脱液存储腔内的洗脱液注入核酸提取腔的操作。Further, when the third mechanical rotary valve is in the sixth position, the second gas source port and the eluent storage chamber port to which the eluent storage chamber belongs, the eluent storage chamber, the third Z-type reagent injection channel, the nucleic acid The extraction chamber, the nucleic acid extraction chamber port, and the atmospheric port are in a connected state; at this time, the second gas source port and the waste liquid chamber port to which the waste liquid chamber belongs, the mixing chamber port to which the mixing chamber belongs, and the first cleaning liquid to which the first cleaning liquid storage chamber belongs The port of the storage chamber and the port of the second cleaning liquid storage chamber to which the second cleaning liquid storage chamber belongs are in an off state. At this time, the external power source provides positive pressure to the second gas source channel, and the operation of injecting the eluent in the eluent storage chamber into the nucleic acid extraction chamber can be realized.
所述卡盒本体底部设置有两个梯形凸台，即第一梯形凸台、第二梯形凸台，用于密封安装裂解管和扩增管。The bottom of the cartridge body is provided with two trapezoidal bosses, namely a first trapezoidal boss and a second trapezoidal boss, which are used for sealingly installing the cracking tube and the amplification tube.
所述卡盒本体顶部设置有三个气源端口，即第一气源端口、第二气源端口、第三气源端口。气源端口作为外接气源接入卡盒的结构，可以接受来自蠕动泵、注射泵、真空发生器生成的气源动力。The top of the cartridge body is provided with three air source ports, namely a first air source port, a second air source port, and a third air source port. The air source port is connected to the cassette as an external air source, and can receive the air source power generated by the peristaltic pump, the syringe pump and the vacuum generator.
所述卡盒本体底部所属第一梯形凸台安装有第二气源通道、第二油封通道和第一裂解液转移通道，插入裂解管内部，分别用于作为液体试剂转移和气源动力提供通道；卡盒本体底部所属第二梯形凸台安装有第二洗脱液转移通道、第四油封通道、第四气源通道；插入扩增管的内部，分别用于作为液体试剂转移和气源动力提供通道。The first trapezoidal boss at the bottom of the cartridge body is provided with a second gas source channel, a second oil seal channel and a first cracking liquid transfer channel, which are inserted into the cracking tube and are respectively used as liquid reagent transfer and gas source power supply channels. The second trapezoidal boss at the bottom of the cartridge body is installed with a second eluent transfer channel, a fourth oil seal channel, and a fourth gas source channel; inserted into the interior of the amplification tube, used for liquid reagent transfer and gas source power respectively Provide access.
所述裂解管和扩增管设置在卡盒本体底部，卡盒本体顶部设置有动力源，用于提供卡盒内部试剂转移所需的动力。The lysis tube and the amplification tube are arranged at the bottom of the cartridge body, and a power source is arranged on the top of the cartridge body to provide the power required for the transfer of reagents inside the cartridge.
所述裂解管用于样本高温裂解，事先预埋裂解液干粉试剂；The lysis tube is used for high-temperature lysis of the sample, and the lysis solution dry powder reagent is pre-buried;
所述扩增管用于核酸吸附和扩增检测，事先预埋扩增液干粉试剂；The amplification tube is used for nucleic acid adsorption and amplification detection, and the amplification liquid dry powder reagent is pre-buried;
所述第一清洗液存储腔预埋有第一清洗液；The first cleaning liquid storage cavity is pre-embedded with the first cleaning liquid;
所述第二清洗液存储腔预埋有第二清洗液。The second cleaning liquid storage cavity is pre-embedded with a second cleaning liquid.
一种封闭式微流控核酸检测卡盒可以用于从生物样本中分离DNA和RNA，并直接在扩增管中完成核酸扩增检测。A closed microfluidic nucleic acid detection cartridge can be used to separate DNA and RNA from biological samples, and complete nucleic acid amplification detection directly in an amplification tube.
进一步地，所述核酸提取和检测步骤包括：Further, the nucleic acid extraction and detection steps include:
步骤1)准备步骤：取临床样本加入一定体积的悬浮液重悬拭子样本；Step 1) Preparation step: take the clinical sample and add a certain volume of suspension to resuspend the swab sample;
步骤2)准备步骤：取一定体积准备步骤中获取的样本注入裂解管，与裂解管中的裂解液干粉试剂充分混合，然后将裂解管安装在封闭式卡盒的第一梯形凸台，在第二梯形凸台预先安装有扩增管；Step 2) Preparation step: Take a certain volume of the sample obtained in the preparation step and inject it into the lysis tube, mix it thoroughly with the lysis solution dry powder reagent in the lysis tube, and then install the lysis tube on the first trapezoidal boss of the closed cartridge. Two trapezoidal bosses are pre-installed with amplification tubes;
步骤3)准备步骤：将卡盒本体放置于一体化核酸检测仪内部，保证裂解管和扩增管对正插入加热模块，完成整个卡盒的正确安装。卡盒本体安放完成之后，确保第一机械旋转阀，第二机械旋转阀，第三机械旋转阀处于第一位置状态，确保各试剂转移通道、气源通道、油封通道保持关闭状态，裂解管和扩增管处于完全密封的状态。Step 3) Preparation steps: place the cartridge body inside the integrated nucleic acid detector, ensure that the lysis tube and the amplification tube are inserted into the heating module in alignment, and complete the correct installation of the entire cartridge. After the cartridge body is installed, make sure that the first mechanical rotary valve, the second mechanical rotary valve, and the third mechanical rotary valve are in the first position, and ensure that each reagent transfer channel, gas source channel, and oil seal channel are kept closed, and the cracking tube and The amplification tube is completely sealed.
步骤4)裂解样本，释放核酸：一体化核酸检测仪中的驱动机构与第一机械旋转阀所属第一转动杆安装处，控制第一机械旋转阀转动到第二位置，外接动力源提供动力给予第一气源端口负压力，即可实现第一油腔中的密封油液转移进入裂解管的操作，密封裂解管中待裂解的样本，防止裂解过程中的气溶胶污染。接着，启动加热过程，使裂解管中的样本被充分地裂解，并尽可能多的释放核酸。Step 4) Lyse the sample and release the nucleic acid: The drive mechanism in the integrated nucleic acid detector and the first rotating rod of the first mechanical rotary valve are installed to control the first mechanical rotary valve to rotate to the second position, and the external power source provides power to give The negative pressure of the first gas source port can realize the operation of transferring the sealed oil in the first oil chamber into the cracking tube, seal the sample to be cracked in the cracking tube, and prevent aerosol pollution during the cracking process. Next, start the heating process so that the sample in the lysis tube is fully lysed and as much nucleic acid is released as possible.
步骤5)转移裂解液进入核酸提取腔：一体化核酸检测仪中的驱动机构与第一机械旋转阀所属第一转动杆安装处，控制第一机械旋转阀转动到第三位置，外接动力源提供动力给予第一气源端口正压力，即可实现裂解管中的裂解液转移进入核酸提取8的操作。Step 5) Transfer the lysate into the nucleic acid extraction chamber: the drive mechanism in the integrated nucleic acid detector and the first rotating rod of the first mechanical rotary valve are installed to control the first mechanical rotary valve to rotate to the third position, and the external power source provides The operation of transferring the lysis solution in the lysis tube into the
步骤6)混合试剂，核酸吸附：一体化核酸检测仪中的驱动机构与第三机械旋转阀所属第三转动杆安装处连接，控制第三机械旋转阀旋转到第二位置，外接动力源提供交替的正压力和负压力给予第二气源通道，即可实现核酸提取腔与混合腔中试剂的混合操作，使预埋在核酸提取腔中的磁珠干粉充分与裂解液混合，以便于充分吸附核酸；Step 6) Mixing reagents, nucleic acid adsorption: The driving mechanism in the integrated nucleic acid detector is connected to the installation of the third rotating rod to which the third mechanical rotary valve belongs, controls the third mechanical rotary valve to rotate to the second position, and the external power source provides alternating The positive pressure and negative pressure are given to the second gas source channel, and the mixing operation of the nucleic acid extraction chamber and the reagents in the mixing chamber can be realized, so that the magnetic bead dry powder pre-buried in the nucleic acid extraction chamber can be fully mixed with the lysis solution, so as to facilitate sufficient adsorption nucleic acid;
步骤7)废液转移步骤一：首先放置磁铁在核酸提取腔的底部。接着，一体化核酸检测仪中的驱动机构与第三机械旋转阀所属第三转动杆安装处连接，控制第三机械旋转阀旋转到第三位置，外接动力源提供负压力给予第二气源通道，即可实现核酸提取腔中废液的转移进入废液腔。Step 7) Waste liquid transfer Step 1: First, place a magnet at the bottom of the nucleic acid extraction chamber. Next, the driving mechanism in the integrated nucleic acid detector is connected to the installation of the third rotating rod belonging to the third mechanical rotary valve, and controls the third mechanical rotary valve to rotate to the third position, and the external power source provides negative pressure to the second air source channel , the waste liquid in the nucleic acid extraction chamber can be transferred into the waste liquid chamber.
步骤8)磁珠清洗步骤A：首先移除磁铁。一体化核酸检测仪中的驱动机构与第三机械旋转阀所属第三转动杆安装处连接，控制第三机械旋转阀旋转到第四位置，外接动力源提供正压力给予第二气源通道，即可实现第一清洗液存储腔内的第一清洗液注入核酸提取腔的操作。随后，重复步骤6)，使清洗液以充分清洗磁珠。Step 8) Magnetic Bead Washing Step A: First remove the magnet. The driving mechanism in the integrated nucleic acid detector is connected to the installation of the third rotating rod belonging to the third mechanical rotary valve, and controls the third mechanical rotary valve to rotate to the fourth position, and the external power source provides positive pressure to the second air source channel, that is, The operation of injecting the first cleaning solution in the first cleaning solution storage chamber into the nucleic acid extraction chamber can be realized. Then, repeat step 6) to make the cleaning solution to wash the magnetic beads sufficiently.
步骤9)废液转移步骤二：重复步骤7)。Step 9) Waste liquid transfer Step 2: Repeat step 7).
步骤10)磁珠清洗步骤B：首先移除磁铁。一体化核酸检测仪中的驱动机构与第三机械旋转阀所属第三转动杆安装处连接，控制第三机械旋转阀旋转到第五位置，外接动力源提供正压力给予第二气源通道，实现第二清洗液存储腔内的第二清洗液注入核酸提取腔的操作。随后，重复步骤6)，使清洗液以充分清洗磁珠。Step 10) Magnetic Bead Washing Step B: First remove the magnet. The driving mechanism in the integrated nucleic acid detector is connected to the installation of the third rotating rod belonging to the third mechanical rotary valve, and controls the third mechanical rotary valve to rotate to the fifth position, and the external power source provides positive pressure to the second air source channel to achieve The operation of injecting the second cleaning solution in the second cleaning solution storage chamber into the nucleic acid extraction chamber. Then, repeat step 6) to make the cleaning solution to wash the magnetic beads sufficiently.
步骤11)废液转移步骤三：重复步骤7)。Step 11) Waste liquid transfer Step 3: Repeat step 7).
步骤12)核酸洗脱步骤：首先移除磁铁。一体化核酸检测仪中的驱动机构与第三机械旋转阀所属第三转动杆安装处连接，控制第三机械旋转阀旋转到第六位置，外接动力源提供正压力给予第二气源通道，即可实现洗脱液存储腔内的洗脱液注入核酸提取腔的操作。随后，重复步骤6)，使洗脱液以充分接触磁珠，与磁珠混合均匀。Step 12) Nucleic acid elution step: First remove the magnet. The driving mechanism in the integrated nucleic acid detector is connected to the installation of the third rotating rod belonging to the third mechanical rotary valve, and controls the third mechanical rotary valve to rotate to the sixth position, and the external power source provides positive pressure to the second air source channel, that is, The operation of injecting the eluate in the eluate storage chamber into the nucleic acid extraction chamber can be realized. Then, repeat step 6) to make the eluent fully contact the magnetic beads and mix with the magnetic beads evenly.
步骤13)转移洗脱液：一体化核酸检测仪中的驱动机构与第二机械旋转阀所属第二转动杆安装处连接，控制第二机械旋转阀旋转到第二位置，外接动力源提供动力给予第三气源端口负压力，即可实现核酸提取腔的洗脱液转移进入扩增管的操作。核酸洗脱液进入扩增管后，与扩增管内的扩增液干粉试剂充分混合，为下游核酸扩增反应做准备。Step 13) Transfer the eluate: the driving mechanism in the integrated nucleic acid detector is connected to the installation of the second rotating rod to which the second mechanical rotary valve belongs, controls the second mechanical rotary valve to rotate to the second position, and the external power source provides power to give The negative pressure of the third gas source port can realize the operation of transferring the eluate of the nucleic acid extraction chamber into the amplification tube. After the nucleic acid eluate enters the amplification tube, it is fully mixed with the amplification liquid dry powder reagent in the amplification tube to prepare for the downstream nucleic acid amplification reaction.
步骤14)扩增反应准备：一体化核酸检测仪中的驱动机构与第二机械旋转阀所属第二转动杆安装处连接，控制第二机械旋转阀旋转到第三位置，外接动力源提供动力给予第三气源端口负压力，实现第二油腔的密封油转移进入扩增管的操作，密封扩增试剂，防止气溶胶污染。Step 14) Amplification reaction preparation: the drive mechanism in the integrated nucleic acid detector is connected to the installation of the second rotating rod to which the second mechanical rotary valve belongs, controls the second mechanical rotary valve to rotate to the third position, and the external power source provides power to give The negative pressure of the third gas source port realizes the operation of transferring the sealing oil of the second oil chamber into the amplification tube, sealing the amplification reagent, and preventing aerosol pollution.
步骤15)扩增检测：一体化核酸检测仪中的驱动机构与第二机械旋转阀所属第二转动杆安装处连接，控制第二机械旋转阀旋转到第一位置，确保扩增管与外界隔绝，防止气溶胶污染。Step 15) Amplification detection: the driving mechanism in the integrated nucleic acid detector is connected to the installation place of the second rotating rod belonging to the second mechanical rotary valve, and the second mechanical rotary valve is controlled to rotate to the first position to ensure that the amplification tube is isolated from the outside world , to prevent aerosol pollution.
本发明相对于现有技术的有益效果是：The beneficial effects of the present invention relative to the prior art are:
本发明提供的一种集成核酸提取、扩增、检测功能于一体的封闭式卡盒，集成了核酸提取和扩增检测两个步骤，在一个小的密闭的盒体内完成，可用于没有分子诊断实验室环境下的核酸提取和扩增检测，具有结构紧凑、易于操作、结构简单、易装配等优点，有效提高分子诊断类产品的环境适应度。The present invention provides a closed cartridge integrating nucleic acid extraction, amplification and detection functions, which integrates two steps of nucleic acid extraction and amplification detection, and is completed in a small closed box, which can be used for molecular diagnosis without molecular diagnosis. Nucleic acid extraction and amplification detection in the laboratory environment has the advantages of compact structure, easy operation, simple structure, and easy assembly, which can effectively improve the environmental adaptability of molecular diagnostic products.
附图说明Description of drawings
图1是本发明的卡盒结构所属第一机械旋转阀、第二机械旋转阀、第三机械旋转阀处于第一位置时的示意图；1 is a schematic diagram of the first mechanical rotary valve, the second mechanical rotary valve, and the third mechanical rotary valve belonging to the cartridge structure of the present invention when they are in a first position;
图2是本发明的卡盒结构所属第一机械旋转阀处于第二位置时的示意图；2 is a schematic view of the first mechanical rotary valve belonging to the cassette structure of the present invention when it is in a second position;
图3是本发明的卡盒结构所属第一机械旋转阀处于第三位置时的示意图；3 is a schematic diagram of the first mechanical rotary valve belonging to the cassette structure of the present invention in a third position;
图4是本发明的卡盒结构所属第三机械旋转阀处于第二位置时的示意图；4 is a schematic diagram of the third mechanical rotary valve belonging to the cassette structure of the present invention when it is in a second position;
图5是本发明的卡盒结构所属第三机械旋转阀处于第三位置时的示意图；5 is a schematic diagram of the third mechanical rotary valve belonging to the cassette structure of the present invention when it is in a third position;
图6是本发明的卡盒结构所属第三机械旋转阀处于第四位置时的示意图；6 is a schematic diagram of the third mechanical rotary valve belonging to the cassette structure of the present invention when it is in a fourth position;
图7是本发明的卡盒结构所属第三机械旋转阀处于第五位置时的示意图；7 is a schematic diagram of the third mechanical rotary valve belonging to the cassette structure of the present invention when it is in a fifth position;
图8是本发明的卡盒结构所属第三机械旋转阀处于第六位置时的示意图；8 is a schematic diagram of the third mechanical rotary valve belonging to the cassette structure of the present invention when it is in a sixth position;
图9是本发明的卡盒结构所属第二机械旋转阀处于第二位置时的示意图；9 is a schematic view of the second mechanical rotary valve belonging to the cassette structure of the present invention when it is in a second position;
图10是本发明的卡盒结构所属第二机械旋转阀处于第三位置时的示意图；10 is a schematic diagram of the second mechanical rotary valve belonging to the cassette structure of the present invention when it is in a third position;
附图标记说明：1为卡盒本体；2为第一梯形凸台；3为第一气源通道；31为第二气源通道；32为第三气源通道；33为第四气源通道；4为裂解管；5为第一裂解液转移通道；51为第二裂解液转移通道；6为第一油封通道；61为第二油封通道；62为第三油封通道；63为第四油封通道；7为废液腔；71为废液腔端口；72为废液转移通道；8为核酸提取腔；81为核酸提取腔端口；9为混合腔；91为混合腔端口；92为混合通道；10为第一洗脱液转移通道；101为第二洗脱液转移通道；11为扩增管；12为第一气源端口；13为第三气源端口；14为第一油腔；141为第一油腔端口；15为第二油腔；151为第二油腔端口；16为第一机械旋转阀；161为第一转动杆安装处；17为第二机械旋转阀；171为第二转动杆安装处；18为第三机械旋转阀；181为第三转动杆安装处；182为大气端口；183为第二气源端口；19为第一阀内油封通道；20为第一阀内气源通道；21为第一阀内裂解液转移通道；22为第一清洗液存储腔；221为第一清洗液存储腔端口；222为第一Z型试剂注入通道；23为第二清洗液存储腔；231为第二清洗液存储腔端口；232为第二Z型试剂注入通道；24为洗脱液存储腔；241为洗脱液存储腔端口；242为第三Z型试剂注入通道；25为第二阀内油封通道；26为第一阀内洗脱液转移通道；27为第二阀内气源通道；28为第二梯形凸台；29为磁铁。Description of reference numerals: 1 is the cartridge body; 2 is the first trapezoidal boss; 3 is the first air source channel; 31 is the second air source channel; 32 is the third air source channel; 33 is the fourth air source channel 4 is the cracking tube; 5 is the first cracking solution transfer channel; 51 is the second cracking solution transfer channel; 6 is the first oil seal channel; 61 is the second oil seal channel; 62 is the third oil seal channel; 63 is the fourth oil seal channel; 7 is a waste liquid chamber; 71 is a waste liquid chamber port; 72 is a waste liquid transfer channel; 8 is a nucleic acid extraction chamber; 81 is a nucleic acid extraction chamber port; 9 is a mixing chamber; 91 is a mixing chamber port; 92 is a mixing channel 10 is the first eluent transfer channel; 101 is the second eluent transfer channel; 11 is the amplification tube; 12 is the first gas source port; 13 is the third gas source port; 14 is the first oil chamber; 141 is the first oil chamber port; 15 is the second oil chamber; 151 is the second oil chamber port; 16 is the first mechanical rotary valve; 161 is the installation of the first rotating rod; 17 is the second mechanical rotary valve; 18 is the third mechanical rotary valve; 181 is the third rotating rod installation; 182 is the atmospheric port; 183 is the second air source port; 19 is the oil seal passage in the first valve; 20 is the first 21 is the first in-valve lysate transfer channel; 22 is the first cleaning solution storage chamber; 221 is the first cleaning solution storage chamber port; 222 is the first Z-type reagent injection channel; 23 is the second Cleaning fluid storage chamber; 231 is the port of the second cleaning fluid storage chamber; 232 is the second Z-type reagent injection channel; 24 is the eluent storage chamber; 241 is the eluent storage chamber port; 242 is the third Z-type reagent injection 25 is the oil seal channel in the second valve; 26 is the eluent transfer channel in the first valve; 27 is the air source channel in the second valve; 28 is the second trapezoidal boss; 29 is a magnet.
具体实施方式Detailed ways
以下结合附图对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.
实施例1Example 1
如图1-2所示，包括卡盒本体1、裂解管4、扩增管11、核酸提取腔8、废液腔7、混合腔9、第一清洗液存储腔22、第二清洗液存储腔23、洗脱液存储腔24、第一油腔14、第二油腔15、第一机械旋转阀16、第二机械旋转阀17、第三机械旋转阀18、第一气源通道3、第二气源通道31、第三气源通道32、第四气源通道33、第一油封通道6、第二油封通道61、第三油封通道62、第四油封通道63、第一裂解液转移通道5、第二裂解液转移通道51、废液转移通道72、混合通道92、第一Z型试剂注入通道222、第二Z型试剂注入通道232、第三Z型试剂注入通242道、第一洗脱液转移通道10、第二洗脱液转移通道101、第一气源端口12、第二气源端口183、第三气源端口13，整体形成一个封闭空间。As shown in Figure 1-2, it includes a cartridge body 1, a
裂解管4可供样本高温裂解使用，其内部预先放置有裂解液干粉试剂。扩增管11可供核酸吸附和扩增检测使用，其内部预先放置有扩增液干粉试剂。第一清洗液存储腔22预埋了70％的乙醇。第二清洗液存储腔23预埋了去离子水。洗脱液存储腔24预埋了洗脱液。The
卡盒本体1在测试前置于一体化核酸检测仪内部，保证裂解管4和扩增管11对正插入加热模块，完成整个卡盒的正确安装。第一机械旋转阀16所属第转动杆安装处161、第二机械旋转阀17所属第转动杆安装处171、第三机械旋转阀18所属第转动杆安装处181可以与一体化核酸检测仪内部的驱动机构连接完成旋转运动，用于切换不同的试剂通道、气源通道、油封。卡盒本体1顶部设置动力源，可以为卡盒内部试剂转移提供所需的动力。The cartridge body 1 is placed inside the integrated nucleic acid detector before the test, to ensure that the
通过旋转第一机械旋转阀16到不同的位置，可以控制第一气源通道3和第二气源通道31、第一油封通道6和第二油封通道62、第一裂解液转移通道5和第二裂解液转移通道51处于关断或开通状态。By rotating the first
通过旋转第二机械旋转阀17到不同的位置，可以控制第一洗脱液转移通道10和第二洗脱液转移通道101、第三油封通道62和第四油封通道63、第三气源通道32和第四气源通道33处于关断或开通状态。By rotating the second
第三机械旋转阀18以核酸提取腔8所属核酸提取腔端口81为中心进行旋转运动，即第三机械旋转阀18所属大气端口182始终与核酸提取腔端口81同心对准。通过第三机械旋转阀18到不同的位置时，可以控制第三机械旋转阀18所属第二气源端口183分别与废液腔7所属废液腔端口71、混合腔9所属废液腔端口91、第一清洗液存储腔22所属第一清洗液存储腔端口221、第二清洗液存储腔23所属第二清洗液存储腔端口231、洗脱液存储腔24所属洗脱液存储腔端口241处于开通或关断状态。The third mechanical
核酸提取和检测时，转动第一机械旋转阀16、第二机械旋转阀17和第三机械旋转阀18到不同的位置，进而导通不同的试剂通道、气路通道、油路通道和各类腔体，然后外接动力源提供动力给卡盒本体1上端的第一气源端口12、第二气源端口183或第三气源端口13，进而控制卡盒本体1内的试剂流动，即将裂解管4中的被裂解的样本转移进入核酸提取腔8，随后在核酸提取腔8内完成磁珠吸附核酸、磁珠清洗、废液转移至废液腔、核酸洗脱、转移核酸至扩增管等核酸提取和检测过程，最后在扩增管11中完成检测。During nucleic acid extraction and detection, turn the first
根据本发明的一个实施例，上述封闭式卡盒具体操作流程如下：According to an embodiment of the present invention, the specific operation process of the above-mentioned closed cartridge is as follows:
1)准备步骤：取临床样本加入一定体积的悬浮液重悬拭子样本；1) Preparation steps: Take clinical samples and add a certain volume of suspension to resuspend the swab samples;
2)准备步骤：取一定体积准备步骤1中获取的样本注入裂解管4，与裂解管4中的裂解液干粉试剂充分混合，然后将裂解管4安装在封闭式卡盒的第一梯形凸台2，在第二梯形凸台28处预先安装有扩增管11；2) Preparation step: Take a certain volume of the sample obtained in the preparation step 1 and inject it into the
3)准备步骤：将卡盒本体1放置于一体化核酸检测仪内部，保证裂解管4和扩增管11对正插入加热模块，完成整个卡盒的正确安装。第一机械旋转阀16所属第转动杆安装处161、第二机械旋转阀17所属第转动杆安装处171、第三机械旋转阀18所属第转动杆安装处181可以与一体化核酸检测仪内部的驱动机构连接。卡盒本体1安放完成之后，确保第一机械旋转阀16，第二机械旋转阀17，第三机械旋转阀18处于第一位置状态，确保各试剂转移通道、气源通道、油封通道保持关闭状态，裂解管4和扩增管11处于完全密封的状态。3) Preparation steps: place the cartridge body 1 inside the integrated nucleic acid detector, ensure that the
4)裂解样本，释放核酸：一体化核酸检测仪中的驱动机构与第一机械旋转阀16所属第一转动杆安装处161，控制第一机械旋转阀16转动到第二位置，第一油封通道6和第二油封通道61，第一气源通道3和第二气源通道31处于开通状态；第一裂解液转移通道5和第二裂解液转移通道51处于关断状态。此时，第一油腔端口141、第一油腔14、第一油封通道6、第一阀内油封通道19、第二油封通61道、裂解管4、第二气源通道31、第一阀内气源通道20、第一气源通道3、第一气源端口12相通。此时，外接动力源提供动力给予第一气源端口12负压力，即可实现第一油腔14中的密封油液转移进入裂解管4的操作，密封裂解管4中待裂解的样本，防止裂解过程中的气溶胶污染。然后，一体化核酸检测仪中的驱动机构与第一机械旋转阀16所属第一转动杆安装处161，控制第一机械旋转阀16转动到第一位置，使第一气源通道3和第二气源通道31、第一油封通道6和第二油封通道61、第一裂解液转移通道5和第二裂解液转移通道51处于关断状态，确保裂解管4完全与外界隔绝。接着，启动加热过程，使裂解管6中的样本被充分地裂解，并尽可能多的释放核酸。4) Lyse the sample and release the nucleic acid: the drive mechanism in the integrated nucleic acid detector and the first
5)转移裂解液进入核酸提取腔：一体化核酸检测仪中的驱动机构与第一机械旋转阀16所属第一转动杆安装处161，控制第一机械旋转阀16转动到第三位置，使第一气源通道3和第二气源通道31、第一裂解液转移通道5和第二裂解液转移通道51处于开通状态；第一油封通道6和第二油封通道61仍然处于关断状态。此时，第一气源端口12、第一气源通道3、第一阀内气源通道20、第二气源通道31、裂解管4、第一裂解管转移通道5、第一阀内裂解液通道21、第二裂解管转移通道51、核酸提取腔8、核酸提取腔端口81、第三机械旋转阀18所属大气端口182连通。此时，外接动力源提供动力给予第一气源端口12正压力，即可实现裂解管4中的裂解液转移进入核酸提取腔8的操作。然后，一体化核酸检测仪中的驱动机构与第一机械旋转阀16所属第一转动杆安装处161，控制第一机械旋转阀16转动到第一位置，使第一气源通道3和第二气源通道31、第一油封通道6和第二油封通道61、第一裂解液转移通道5和第二裂解液转移通道51处于关断状态，确保裂解管4完全与外界隔绝。5) Transfer the lysate into the nucleic acid extraction chamber: the drive mechanism in the integrated nucleic acid detector and the first
6)混合试剂，核酸吸附：一体化核酸检测仪中的驱动机构与第三机械旋转阀18所属第三转动杆安装处181连接，控制第三机械旋转阀18旋转到第二位置，第二气源端口183与混合腔9所属混合腔端口91、混合腔9、混合通道92、核酸提取腔8、核酸提取腔端口81、第三机械旋转阀18所属大气端口182处于连通状态；此时，第二气源端口183与废液腔7所属废液腔端口71、第一清洗液存储腔22所属第一清洗液存储腔端口221、第二清洗液存储腔23所属第二清洗液存储腔端口231、洗脱液存储腔24所属洗脱液存储腔端口241处于关断状态。此时，外接动力源提供交替的正压力和负压力给予第二气源通道183，即可实现核酸提取腔8与混合腔9中试剂的混合操作，使预埋在核酸提取腔8中的磁珠干粉充分与裂解液混合，以便于充分吸附核酸；6) Mixed reagents, nucleic acid adsorption: The drive mechanism in the integrated nucleic acid detector is connected to the third
7)废液转移步骤1：首先放置磁铁29在核酸提取腔8的底部。接着，一体化核酸检测仪中的驱动机构与第三机械旋转阀18所属第三转动杆安装处181连接，控制第三机械旋转阀18旋转到第三位置，第二气源端口183与废液腔7所属废液腔端口71、废液腔、废液转移通道7、核酸提取腔8、核酸提取腔端口81、大气端口182处于连通状态；此时，第二气源端口183与混合腔9所属混合腔端口91、第一清洗液存储腔22所属第一清洗液存储腔端口221、第二清洗液存储腔23所属第二清洗液存储腔端口231、洗脱液存储腔24所属洗脱液存储腔端口241处于关断状态。此时，外接动力源提供负压力给予第二气源通道183，即可实现核酸提取腔8中废液的转移进入废液腔7。7) Waste liquid transfer step 1: First, place the
8)磁珠清洗步骤1：首先移除磁铁29。一体化核酸检测仪中的驱动机构与第三机械旋转阀18所属第三转动杆安装处181连接，控制第三机械旋转阀18旋转到第四位置，第二气源端口183与第一清洗液存储腔22所属第一清洗液存储腔端口221、第一清洗液存储腔22、第一Z型试剂注入通道222、核酸提取腔8、核酸提取腔端口81、大气端口182处于连通状态；此时，第二气源端口183与废液腔7所属废液腔端口71、混合腔9所属混合腔端口91、第二清洗液存储腔23所属第二清洗液存储腔端口231、洗脱液存储腔24所属洗脱液存储腔端口241处于关断状态。此时，外接动力源提供正压力给予第二气源通道183，即可实现第一清洗液存储腔22内的第一清洗液注入核酸提取腔8的操作。随后，重复第6步骤中的内容，使清洗液以充分清洗磁珠。8) Magnetic bead cleaning step 1: First remove the
9)废液转移步骤2：重复第7步骤中的内容。9) Waste liquid transfer step 2: Repeat the content in
10)磁珠清洗步骤2：首先移除磁铁29。一体化核酸检测仪中的驱动机构与第三机械旋转阀18所属第三转动杆安装处181连接，控制第三机械旋转阀18旋转到第五位置，第二气源端口183与第二清洗液存储腔23所属第二清洗液存储腔端口231、第二清洗液存储腔23、第二Z型试剂注入通道232、核酸提取腔8、核酸提取腔端口81、大气端口182处于连通状态；此时，第二气源端口183与废液腔7所属废液腔端口71、混合腔9所属混合腔端口91、第一清洗液存储腔22所属第一清洗液存储腔端口221、洗脱液存储腔24所属洗脱液存储腔端口241处于关断状态。此时，外接动力源提供正压力给予第二气源通道183，即可实现第二清洗液存储腔23内的第二清洗液注入核酸提取腔8的操作。随后，重复第6步骤中的内容，使清洗液以充分清洗磁珠。10) Magnetic bead cleaning step 2: First remove the
11)废液转移步骤3：重复第7步骤中的内容。11) Waste liquid transfer step 3: Repeat the content in
12)核酸洗脱步骤：首先移除磁铁29。一体化核酸检测仪中的驱动机构与第三机械旋转阀18所属第三转动杆安装处181连接，控制第三机械旋转阀18旋转到第六位置，第二气源端口183与洗脱液存储腔24所属洗脱液存储腔端口241、洗脱液存储腔24、第三Z型试剂注入通道242、核酸提取腔8、核酸提取腔端口81、大气端口182处于连通状态；此时，第二气源端口183与废液腔7所属废液腔端口71、混合腔9所属混合腔端口91、第一清洗液存储腔22所属第一清洗液存储腔端口221、第二清洗液存储腔23所属第二清洗液存储腔端口231处于关断状态。此时，外接动力源提供正压力给予第二气源通道183，即可实现洗脱液存储腔24内的洗脱液注入核酸提取腔8的操作。随后，重复第6步骤中的内容，使洗脱液以充分接触磁珠，与磁珠混合均匀。在进行下一步操作之前，一体化核酸检测仪中的驱动机构与第三机械旋转阀18所属第三转动杆安装处181连接，控制第三机械旋转阀18旋转到第一位置，此种状态下，第二气源端口183与废液腔7所属废液腔端口71、混合腔9所属废液腔端91口、第一清洗液存储腔22所属第一清洗液存储腔端口221、第二清洗液存储腔23所属第二清洗液存储腔端口231、洗脱液存储腔24所属洗脱液存储腔端口241处于关断状态。12) Nucleic acid elution step: the
13)转移洗脱液：一体化核酸检测仪中的驱动机构与第二机械旋转阀17所属第二转动杆安装处171连接，控制第二机械旋转阀17旋转到第二位置，第一洗脱液转移通道10和第二洗脱液转移通道101、第三气源通道32和第四气源通道33处于开通状态；第三油封通道62和第四油封通道63仍然处于关断状态。此时，第三气源端口13、第三气源通道32、第二阀内气源通道27、第四气源通道33、扩增管11、第二洗脱液转移通道101、第一阀内洗脱液转移通道26、第一洗脱液转移通道10、核酸提取腔8、核酸提取腔端口81、第三机械旋转阀18所属大气端口182连通。此时，外接动力源提供动力给予第三气源端口13负压力，即可实现核酸提取腔8的洗脱液转移进入扩增管11的操作。核酸洗脱液进入扩增管11后，与扩增管11内的扩增液干粉试剂充分混合，为下游核酸扩增反应做准备。13) Transfer the eluate: the drive mechanism in the integrated nucleic acid detector is connected to the second
14)扩增反应准备：一体化核酸检测仪中的驱动机构与第二机械旋转阀17所属第二转动杆安装处171连接，控制第二机械旋转阀17旋转到第三位置，第三油封通道62和第四油封通道63，第三气源通道32和第四气源通道33处于开通状态；第一洗脱液转移通道10和第二洗脱液转移通道101处于关断状态。此时，第三气源端口13、第三气源通道32、第二阀内气源通道27、第四气源通道33、扩增管11、第四油封通道63、第二阀内油封通道25、第三油封通道62、第二油腔15、第二油腔端口151相互连通。此时，外接动力源提供动力给予第三气源端口13负压力，即可实现第二油腔15的密封油转移进入扩增管11的操作，密封扩增试剂，防止气溶胶污染。14) Preparation of amplification reaction: the driving mechanism in the integrated nucleic acid detector is connected to the second
15)扩增检测：一体化核酸检测仪中的驱动机构与第二机械旋转阀17所属第二转动杆安装处171连接，控制第二机械旋转阀17旋转到第一位置，第三油封通道62和第四油封通道63，第三气源通道32和第四气源通道33，第一洗脱液转移通道10和第二洗脱液转移通道101处于关断状态，确保扩增管11与外界隔绝，防止气溶胶污染。15) Amplification detection: the driving mechanism in the integrated nucleic acid detector is connected to the second
本发明的一种集成核酸提取、扩增、检测功能于一体的封闭式微流控核酸检测卡盒，可用于没有分子诊断实验室环境下的核酸提取和扩增检测，具有结构紧凑、易于操作、环境容忍度高、可检测多个指标等优点。A closed microfluidic nucleic acid detection cartridge integrating nucleic acid extraction, amplification and detection functions of the present invention can be used for nucleic acid extraction and amplification detection in laboratory environments without molecular diagnosis, and has the advantages of compact structure, easy operation, It has the advantages of high environmental tolerance and can detect multiple indicators.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still understand the foregoing embodiments. The technical solutions described are modified, or some technical features thereof are equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
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