CN107603866B - Microfluidic chip kit for detecting 10 respiratory tract infection pathogens and use method thereof - Google Patents
Microfluidic chip kit for detecting 10 respiratory tract infection pathogens and use method thereof Download PDFInfo
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Abstract
The invention provides a micro-fluidic chip kit for detecting 10 respiratory tract infection pathogens and a using method thereof, the kit combines a Taqman probe fluorescence PCR technology with a micro-fluidic chip, detects 10 common respiratory tract infection pathogens, can obtain a detection result within 2 hours, and has strong specificity and sensitivity of 100 copies/mu L; comprises a sample introduction chamber, a reaction chamber and a microfluidic flow channel; the reaction chambers at least comprise 10 reaction chambers, each reaction chamber is mutually independent and is pre-provided with reagent dry powder for amplifying a respiratory tract pathogen, and the reagent dry powder comprises a primer and a TaqMan probe for amplifying the corresponding respiratory tract pathogen; the reagent dry powder preset in each reaction chamber can amplify any one of 10 respiratory pathogens respectively, and the respiratory pathogens amplified by the reagent dry powder in all the reaction chambers can encapsulate the 10 respiratory pathogens.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a kit for detecting a micro-fluidic chip for detecting 10 respiratory tract infection pathogens and a using method thereof.
Background
Respiratory tract infection is one of the most common diseases worldwide, the incidence rate is predominant in the overall structure of the incidence rate of residents in various countries, and about 10% of residents suffer from respiratory tract infection at the peak of the annual flow rate. Respiratory tract infection can cause rhinitis, nasal discharge, nasal obstruction, cough, mild pharyngitis, systemic fever and other symptoms, and can cause dyspnea and even death in severe cases. Acute respiratory tract infection has become one of the important infectious diseases of children at home and abroad, and the incidence rate of the acute respiratory tract infection is the first of various diseases of children. Pathogens causing respiratory infections in children mainly include viruses, bacteria, mycoplasma, chlamydia and the like, and rapid and accurate diagnosis and appropriate treatment are important prerequisites for reducing hospitalization of children and unnecessary use of antibacterial drugs. Therefore, the rapid detection of respiratory pathogens is of great significance for early clinical diagnosis.
Microfluidic chips or Lab-on-a-chips refer to a technology for integrating basic operation units such as sample preparation, biological and chemical reaction, separation, detection, and the like, which are related in the fields of biology, chemistry, and the like, or basically on a Chip with a few square centimeters (even smaller) to complete different biological or chemical reaction processes and analyze products thereof. This technique is in principle suitable for the reaction, separation and detection of various types of molecules ranging from nucleic acids, proteins up to small organic and inorganic molecules. The micro-fluidic chip has the characteristics of controllable liquid flow, extremely less consumption of samples and reagents, ten-fold or hundred-fold improvement of analysis speed and the like, can simultaneously analyze hundreds of samples in a few minutes or even shorter time, and can realize the whole processes of pretreatment and analysis of the samples on line.
The detection methods aiming at respiratory tract infection pathogens in the current market mainly comprise a fluorescence PCR method, an immunization method and a bacterial culture method. The fluorescence PCR method is to monitor the PCR process in real time through a fluorescence signal in the PCR amplification process and qualitatively or quantitatively detect a target gene; the immunization method is to detect target protein through specific combination of antigen and antibody; the bacterial culture method is to culture pathogen bacteria on a specific culture medium and observe and detect the generated bacterial colonies.
Although the common fluorescent PCR detection reagent has better sensitivity and specificity, the common fluorescent PCR detection reagent mostly aims at single or a plurality of pathogens, and a plurality of different detection reagent kits are matched to detect a plurality of pathogens, so that the operation is inconvenient and the cost is higher; the immunity method detection reagent can detect a plurality of pathogens simultaneously, but because the immunity method detects the specific antibodies generated by organisms caused by the pathogens in the samples, a longer blank window period exists, and the sensitivity is relatively lower; for the detection of bacterial pathogens, the bacterial culture method has high accuracy, but has long time consumption, poor culture effect, and a lot of bacteria can not be cultured, so that the optimal treatment time is delayed.
Disclosure of Invention
The invention provides a kit for detecting a microfluidic chip and a using method thereof aiming at 10 common respiratory infection pathogens, including mycoplasma pneumoniae, chlamydia pneumoniae, legionella pneumophila, bordetella pertussis, rhinovirus, influenza A virus, influenza B virus, respiratory adenovirus, syncytial virus, parainfluenza virus and the like, so as to realize the detection requirement of simultaneously and rapidly detecting 10 respiratory infection pathogens.
In order to achieve the purpose, the invention adopts the following technical scheme:
a micro-fluidic chip kit for detecting 10 respiratory tract infection pathogens comprises a kit body, wherein the kit body comprises a sample introduction chamber, a reaction chamber and a micro-fluidic flow channel for realizing the communication of the sample introduction chamber and the reaction chamber;
the reaction chambers at least comprise 10 reaction chambers, each reaction chamber is mutually independent, and each reaction chamber is pre-provided with reagent dry powder for amplifying a respiratory tract pathogen, and the reagent dry powder comprises a primer and a TaqMan probe for amplifying the corresponding respiratory tract pathogen;
the reagent dry powder preset in each reaction chamber can amplify any one of the following 10 respiratory pathogens, and meanwhile, the respiratory pathogens amplified by the reagent dry powder in all the reaction chambers can include the following 10 respiratory pathogens:
mycoplasma pneumoniae (Mycoplasma pneumoniae), chlamydia pneumoniae (chlamydophila), Legionella pneumophila (Legionella pneumophila), bordetella pertussis (bordethouse), rhinoviruses (Human rhinoviruses), respiratory adenoviruses (Adenovirus), Influenza a viruses (inflenza a viruses), Influenza B viruses (Influenza B viruses), respiratory syncytial viruses (respiratory syncytial viruses), and parainfluenza viruses (Human parainfluenza viruses);
the sample introduction cavity is arranged at the middle position of the kit body and is provided with a sample introduction hole, a sample outlet, an air inlet hole and an air exhaust hole;
the micro-fluidic runner includes and advances kind sprue, advances kind subchannel, exhaust sprue, exhaust subchannel and overflow channel, wherein: the number of the sample feeding branch channels, the exhaust branch channels and the overflow channels is consistent with that of the reaction chambers;
the sample outlet of the sample injection cavity is communicated with one end of a sample injection main flow channel, the other end of the sample injection main flow channel is provided with sample branch flow outlets the number of which is consistent with that of the reaction cavities, and the sample branch flow outlets of the sample injection main flow channel are communicated with the sample injection ports of the corresponding reaction cavities one by one through sample injection branch flow channels; each sample feeding branch channel is provided with a sample feeding one-way valve;
the air inlet of the sample introduction cavity is communicated with one end of the exhaust main flow channel, the other end of the exhaust main flow channel is provided with exhaust gathering ports with the number consistent with that of the reaction cavities, and each exhaust gathering port of the exhaust main flow channel is communicated with the air inlet of the corresponding reaction cavity one by one sequentially through an exhaust branch channel, an exhaust one-way valve, a breathable water-blocking filter element and an overflow channel.
As a further improvement of the invention, the sequence of the primer is as follows: mycoplasma pneumoniae (Mycoplasma pneumoniae), SEQ ID nos. 1-2; chlamydia pneumoniae (Chlamydophila pneumoniae), SEQ ID nos. 4 to 5; legionella pneumophila (Legionella pneumophila), SEQ ID No. 7-8; bordetella pertussis (Bordetella pertussis), SEQ ID No. 10-11; rhinoviruses (Human rhinoviruses), SEQ ID NO. 13-14; respiratory Adenovirus (Adenovirus), SEQ ID nos. 16-17; influenza a virus (Influenza Avirus), SEQ ID No. 19-20; influenza B virus (Influenza B virus), SEQ ID NO. 22-23; respiratory syncytial virus (respiratory syncytial virus), SEQ ID nos. 25 to 26; parainfluenza virus (human parainfluenza virus), SEQ ID Nos. 28 to 29;
the sequence of the TaqMan probe is as follows: mycoplasma pneumoniae (Mycoplasma pneumoniae), SEQ ID No. 3; chlamydia pneumoniae (Chlamydophila pneumoniae), SEQ ID No. 6; legionella pneumophila (legionella pneumophila), SEQ ID No. 9; bordetella pertussis (Bordetella pertussis), SEQ ID No. 12; rhinovirus (Human rhinovirus), SEQ ID No. 15; respiratory Adenovirus (Adenovirus), SEQ ID No. 18; influenza a virus (Influenza Avirus), SEQ ID No. 21; influenza B virus (Influenza B virus), SEQ id No. 24; respiratory syncytial virus (respiratory syncytial virus), SEQ ID No. 27; parainfluenza virus (human parainfluenza virus), SEQ ID NO. 30.
As a further improvement of the invention, the final concentration of the primer in the amplification system is 100-1000 nM; the final concentration of the TaqMan probe in an amplification system is 50-500 nM.
As a further improvement of the invention, an RT-PCR buffer, a mixed enzyme solution and a positive quality control product are preset in the sample introduction chamber.
As a further improvement of the invention, the RT-PCR buffer comprises PCR buffer, dATP, dUTP, dCTP, dGTP and MgCl2。
As a further improvement of the invention, the mixed enzyme solution comprises HotStart Taq enzyme, reverse transcriptase and UNG enzyme.
As a further improvement of the invention, the positive quality control product contains plasmids corresponding to the 10 respiratory tract pathogen amplification gene sequences.
As a further improvement of the invention, the kit body is of a three-piece structure and comprises an upper-layer membrane, a middle-layer membrane and a bottom-layer membrane which are hermetically bonded from top to bottom in sequence; wherein:
the sample introduction cavity comprises a sample introduction groove, and a sample introduction hole and an exhaust hole of the sample introduction cavity are through holes which penetrate through the upper layer membrane and the middle layer membrane and are communicated with the reaction groove; the reaction chamber comprises a reaction tank;
the sample injection main flow passage and the exhaust main flow passage are respectively communicated with the sample injection groove, the sample injection main flow passage and the exhaust main flow passage are all arranged on the bottom layer membrane, and the sample injection main flow passage and the exhaust main flow passage are both T-shaped flow passages and are respectively arranged at two sides of the sample injection groove;
the T-shaped flow channel comprises a transverse straight section flow channel and a vertical section flow channel, the transverse straight section flow channel is arranged in the middle of the bottom membrane and is communicated with the sample feeding groove, and the vertical section flow channel is arranged along the side edge of the bottom membrane;
the various branch flow outlets of the sample adding main flow channel are arranged on the vertical section flow channel of the sample adding main flow channel, and the exhaust convergence ports of the exhaust main flow channel are arranged on the vertical section flow channel of the exhaust main flow channel;
the reaction tanks are arranged on the bottom-layer diaphragm and are positioned on the inner side of the vertical section runner of the main sampling runner, and meanwhile, the reaction tanks are symmetrically distributed on two sides of the horizontal straight section runner of the main sampling runner;
the sample injection sub-channel, the exhaust sub-channel and the overflow channel are respectively arranged on the middle layer chip, wherein:
the outlet of each sample feeding branch channel is communicated with the corresponding reaction tank through a corresponding sample feeding guide hole;
the inlet of each overflow channel is communicated with the corresponding reaction tank through the corresponding overflow guide liquid inlet hole, and the outlet of each overflow channel is communicated with the liquid inlet of the corresponding exhaust one-way valve through the corresponding overflow guide liquid outlet hole;
the outlet of each exhaust one-way valve is connected with the inlet of an exhaust branch channel through a corresponding exhaust guide hole, the outlet of each exhaust branch channel is communicated with the inlet of a corresponding air-permeable water-blocking filter element, and the outlet of each air-permeable water-blocking filter element is communicated with an exhaust gathering port corresponding to an exhaust main channel;
the exhaust one-way valves are arranged on the bottom-layer membrane and are positioned on the inner side of the vertical section flow channel of the exhaust main flow channel, and meanwhile, the exhaust one-way valves are symmetrically distributed on two sides of the transverse straight section flow channel of the exhaust main flow channel;
the sample adding one-way valves are arranged on the middle layer chip, and the positions of the sample adding one-way valves on the middle layer chip correspond to the positions of the various branch outlets of the sample adding main flow channel one by one;
the sample adding guide hole, the overflow guide liquid inlet hole, the overflow guide liquid outlet hole and the exhaust guide hole are respectively vertically arranged on the middle layer chip in a penetrating way;
the breathable water-blocking filter cores are arranged on the middle chip, and the positions of the breathable water-blocking filter cores on the middle chip are in one-to-one correspondence with the positions of the exhaust gathering ports of the exhaust main flow channel.
As a further improvement of the invention, the exhaust one-way valve comprises a diaphragm, one end of the diaphragm is pressed between the middle diaphragm and the bottom diaphragm, the other end of the diaphragm is suspended, and the bottom diaphragm is provided with a diaphragm caulking groove at a position corresponding to the diaphragm suspension end; the diaphragm suspension end is opposite to the exhaust guide hole, the size of the outer edge of the diaphragm suspension end is larger than the aperture of the exhaust guide hole and smaller than the size of the inner edge of the diaphragm caulking groove, and meanwhile, a gap which only allows gas to pass through is formed between the outer edge of the diaphragm suspension end and the inner wall of the diaphragm caulking groove.
Another technical object of the present invention is to provide a method for using the above microfluidic chip kit for detecting 10 respiratory tract infection pathogens, which comprises the following steps:
(1) diluting a sample DNA/RNA co-extraction template by RNase-free water;
(2) injecting sample into the diluted sample DNA/RNA co-extraction template through a sample injection hole of a sample injection chamber, mixing the sample DNA/RNA co-extraction template with a mixed enzyme solution and an RT-PCR buffer which are preset in the sample injection chamber, sequentially passing through a sample injection main flow channel, a sample injection one-way valve and a sample injection sub-flow channel, simultaneously injecting sample into each reaction chamber, and stopping sample injection until the overflow channel is filled with the sample flowing into each reaction chamber; at the moment, under the combined action of the exhaust check valve and the sample adding check valve, the whole reaction chamber is in a liquid interception state, and the reaction chamber is only in a gas passing state at the rear output end;
(3) carrying out PCR amplification reaction on the sample in the reaction chamber and reagent dry powder preset in the reaction chamber;
the conditions of the PCR amplification reaction are as follows: reverse transcription at 50 deg.C for 15-30 min; pre-denaturation at 92-97 deg.C for 1-10 min; denaturation at 92-97 deg.C for 10-15 s; annealing at 58-62 deg.C for 35-50 s; 40-45 cycles.
Has the advantages that:
the invention provides a microfluidic chip kit, which is provided with more than 10 reaction chambers, and through designing a special microfluidic flow channel and installing a sample adding one-way valve and an exhaust one-way valve on the microfluidic flow channel, when each reaction chamber is used for adding samples, the samples can be added to a plurality of reaction chambers through one sample adding chamber; after the sample adding is finished, the reaction chambers can be mutually independent in PCR amplification reaction through the control of the sample adding one-way valve and the exhaust one-way valve, so that the reaction chambers are not polluted or interfered with each other, and no amplification product is leaked after the detection is finished; in addition, one of 10 common respiratory tract pathogen PCR amplification reagent dry powders is respectively preset in each reaction chamber, so that whether a sample added from a sample adding chamber has the 10 respiratory tract pathogens can be detected at one time by using the microfluidic chip kit, and the method is rapid and convenient;
the kit disclosed by the invention combines a Taqman probe fluorescence PCR technology with a microfluidic chip, detects 10 common respiratory infection pathogens, can obtain a detection result within 2 hours, and is strong in specificity and high in sensitivity which can reach 100 copies/mu L.
The invention adopts a UNG enzyme/dUTP anti-pollution system, and can reduce pollution interference caused by the product of the previous PCR reaction.
According to the invention, the reverse transcriptase and the HotStart Taq enzyme are mixed for use, the reverse transcription process is firstly carried out to synthesize cDNA, then PCR amplification is carried out, no random primer is added, the process is completely completed by an instrument, the experimental operation steps can be reduced, the time is saved, and the possible sample pollution is avoided.
The probe used in the invention is a 5' end FAM-labeled TaqMan probe, and the two ends of the probe are respectively labeled with oligonucleotide of a fluorescence reporter group (R) and a fluorescence quenching group (Q). When the probe is complete, namely in a random state and a non-PCR product hybridization state, the fluorescence emitted by the reporter group is absorbed by the quencher group. In the fluorescent PCR amplification process, when a specific PCR product and a TaqMan probe are subjected to hybridization reaction, the 5' end exonuclease activity of the HotStart Taq enzyme is also cracked at the same time, and the fluorescence released by the reporter group can be detected by a fluorimeter arranged in a quantitative detector. After each cycle of PCR, the fluorescent signal has a synchronous exponential growth process as the target fragment, and the strength of the fluorescent signal represents the copy number of the template RNA. Therefore, the invention can be used for simple qualitative detection and quantitative detection of specific content of the sample.
In conclusion, compared with the common fluorescence PCR method, the immunization method, the bacterial culture method and the like, the invention has the following advantages:
1. the specificity is strong: the primer probe is designed aiming at specific conserved region sequences of 10 common respiratory tract infection pathogens, and has strong specificity.
2. The sensitivity is high: the invention can detect the target gene sequence with the concentration of 100 copies/mu L.
3. The flux is high: the invention can simultaneously detect 10 common respiratory tract infection pathogens.
4. The detection process is a closed tube reaction, and the reverse transcription process and the PCR amplification process are combined, so that the experimental steps are reduced, and the possibility of pollution and result deviation is greatly reduced.
5. The operation is simple and quick, and the result obtained from the sample submission can be finished within 3 hours.
6. The result is clearly interpreted and objectively; the results can also be quantitatively analyzed if desired.
7. Safety: the whole system does not contain toxic and harmful substances, does not need post-treatment of PCR products, and is harmless to operators and environment.
Drawings
FIG. 1 shows a schematic diagram of the overall structure of a microfluidic chip for nucleic acid amplification detection according to the present invention.
Fig. 2 shows a schematic view of the structure of the bottom layer film sheet.
Fig. 3 shows a schematic view of the structure of the middle layer membrane.
Fig. 4 shows a schematic structural diagram of the upper membrane.
FIG. 5 shows a schematic view of the installation of the vent check valve on the bottom membrane.
Fig. 6 shows a schematic view of the structure of the exhaust check valve.
Fig. 7 shows a schematic view of the structure of the membrane.
Among them are:
10. an upper membrane sheet;
11. a first sample adding hole; 12. a first exhaust hole; 13. a sample introduction sub-channel; 14. an overflow channel; 15. an exhaust gas bypass;
20. a middle layer membrane;
21. a second sampling hole; 22. a second exhaust hole; 23. a sample adding one-way valve; 24. a sample application guide hole; 25. an overflow guide hole I; 26. a second overflow guide hole; 27. an exhaust guide hole; 28. a breathable water-blocking filter element;
30. a bottom layer membrane;
31. a first sample adding main flow channel; 32. a second sample injection main flow channel; 33. a reaction chamber; 34. a first exhaust main flow channel; 35. a second exhaust main flow channel; 36. an exhaust check valve;
40. a membrane; 41. a diaphragm connecting end; 42. a diaphragm suspension end; 43. a diaphragm suspension end gap; 44. and (4) embedding the diaphragm into the groove.
Detailed Description
As shown in fig. 1, the microfluidic chip kit for detecting 10 respiratory tract infection pathogens of the present invention comprises a kit body, wherein the kit body comprises a sample introduction chamber, a reaction chamber, and a microfluidic channel for communicating the sample introduction chamber and the reaction chamber; the reaction chambers at least comprise 10 reaction chambers, each reaction chamber is mutually independent, and each reaction chamber is pre-provided with reagent dry powder for amplifying a respiratory tract pathogen, and the reagent dry powder comprises a primer and a TaqMan probe for amplifying the corresponding respiratory tract pathogen;
the reagent dry powder preset in each reaction chamber can amplify any one of the following 10 respiratory pathogens, and meanwhile, the respiratory pathogens amplified by the reagent dry powder in all the reaction chambers can include the following 10 respiratory pathogens:
mycoplasma pneumoniae (Mycoplasma pneumoniae), chlamydia pneumoniae (chlamydophila), Legionella pneumophila (Legionella pneumophila), bordetella pertussis (bordethouse), rhinovirus (Human rhinovirus), respiratory Adenovirus (Adenovirus), Influenza a (inflza Avirus), Influenza B virus (Influenza B virus), respiratory syncytial virus (respiratory syncytial virus), and parainfluenza virus (Human parainfluenza virus);
the sample introduction cavity is arranged at the middle position of the kit body and is provided with a sample introduction hole, a sample outlet, an air inlet hole and an air exhaust hole;
the micro-fluidic runner includes and advances kind sprue, advances kind subchannel, exhaust sprue, exhaust subchannel and overflow channel, wherein: the number of the sample feeding branch channels, the exhaust branch channels and the overflow channels is consistent with that of the reaction chambers;
the sample outlet of the sample injection cavity is communicated with one end of a sample injection main flow channel, the other end of the sample injection main flow channel is provided with sample branch flow outlets the number of which is consistent with that of the reaction cavities, and the sample branch flow outlets of the sample injection main flow channel are communicated with the sample injection ports of the corresponding reaction cavities one by one through sample injection branch flow channels; each sample feeding branch channel is provided with a sample feeding one-way valve;
the air inlet of the sample introduction cavity is communicated with one end of the exhaust main flow channel, the other end of the exhaust main flow channel is provided with exhaust gathering ports with the number consistent with that of the reaction cavities, and each exhaust gathering port of the exhaust main flow channel is communicated with the air inlet of the corresponding reaction cavity one by one sequentially through an exhaust branch channel, an exhaust one-way valve, a breathable water-blocking filter element and an overflow channel.
The kit body disclosed by the embodiment of the invention is of a three-piece structure and comprises three layers of membranes, wherein the three layers of membranes are hermetically bonded; preferably, the sealing is formed into a whole body by thermal bonding or ultrasonic bonding, etc., so as to meet the requirements of nucleic acid amplification and detection.
The three layers of membranes are all preferably made of transparent hard materials, and the transparent hard materials are preferably made of one of high molecular polymers, quartz glass, silicon wafers and the like.
The three layers of membranes are an upper membrane 10, a middle membrane 20 and a bottom membrane 30 from top to bottom in sequence.
The specific structure of each membrane is described in detail below.
As shown in fig. 2, the bottom layer membrane 30 is provided with a sample inlet channel, a sample feeding main channel, an exhaust main channel, a plurality of reaction channels 33, and exhaust check valves 36 with the same number as the reaction channels; wherein: the sample introduction cavity comprises a sample introduction groove, and a sample introduction hole and an exhaust hole of the sample introduction cavity are through holes which penetrate through the upper layer membrane and the middle layer membrane and are communicated with the reaction groove; the reaction chamber comprises a reaction tank;
the sample adding main flow channel and the exhaust main flow channel are respectively communicated with the sample feeding groove, the sample adding main flow channel is a T-shaped flow channel formed by a first sample adding main flow channel 31 and a second sample adding main flow channel 32, and the exhaust main flow channel is a T-shaped flow channel formed by a first exhaust main flow channel 34 and a second exhaust main flow channel 35; the first sample injection main flow channel 31 and the first exhaust main flow channel 34 are transverse straight section flow channels of the T-shaped flow channel, and the second sample injection main flow channel 32 and the second exhaust main flow channel 35 are vertical section flow channels of the T-shaped flow channel.
The second sample adding main flow channel is arranged along one length side of the bottom layer membrane, namely the second sample adding main flow channel is parallel to the length side of the bottom layer membrane.
The first sample adding main flow channel is arranged in the middle of the bottom membrane, the first sample adding main flow channel is perpendicular to the second sample adding main flow channel, and an outlet of the first sample adding main flow channel is communicated with the second sample adding main flow channel.
The reaction tanks are arranged on two sides of the first sample adding main flow channel in parallel, the reaction tanks are preferably parallel to each other and are located on the same straight line, and the straight line where the reaction tanks are located is parallel to the sample adding main flow channel.
The first exhaust main flow channel is arranged along the other length side of the bottom membrane, namely the first exhaust main flow channel is parallel to the length side of the bottom membrane.
In the invention, the first sample injection main flow channel and the second exhaust main flow channel are preferably coaxially arranged.
The second exhaust main flow channel is also arranged in the middle of the bottom membrane and is vertical to the first exhaust main flow channel, and an inlet of the second exhaust main flow channel is communicated with the first exhaust main flow channel.
The exhaust check valves are arranged on two sides of the exhaust main flow channel II in parallel, each exhaust check valve corresponds to one reaction tank, and the reaction tanks are preferably arranged coaxially with the corresponding exhaust check valves.
The exhaust one-way valve can block water but enable gas to pass through, as shown in fig. 5, 6 and 7, the exhaust one-way valve comprises a diaphragm, one end of the diaphragm is pressed between a middle diaphragm and a bottom diaphragm, the other end of the diaphragm is suspended, and a diaphragm caulking groove is formed in the bottom diaphragm at a position corresponding to the suspended end of the diaphragm; the diaphragm suspension end is opposite to the exhaust guide hole, the size of the outer edge of the diaphragm suspension end is larger than the aperture of the exhaust guide hole and smaller than the size of the inner edge of the diaphragm caulking groove, and meanwhile, a gap which only allows gas to pass through is formed between the outer edge of the diaphragm suspension end and the inner wall of the diaphragm caulking groove.
In order to facilitate installation, the diaphragms of the exhaust check valve are integrated, specifically, the diaphragms are integrally pressed at the ends of the middle diaphragm and the bottom diaphragm to form a diaphragm connecting end 41, the diaphragm suspension ends 42 are equidistantly arranged at the diaphragm connecting end 41 at intervals, a diaphragm suspension end gap 43 exists between every two adjacent diaphragm suspension ends 42, each diaphragm suspension end gap 43 only allows gas to pass through, and liquid water molecules cannot pass through.
The diaphragm is preferably made of elastic membrane material, and the cross section of the suspension end of the diaphragm is preferably rectangular.
When liquid water molecules pass through the exhaust guide hole from the exhaust branch channel and enter the inlet of the exhaust check valve, the diaphragm suspension end can be opened by the pressure of the liquid water molecules, so that the liquid water molecules normally pass through. When gas passes through the exhaust branch channel or the inlet of the exhaust one-way valve, the gas can pass through the gap of the diaphragm suspension end, and both directions of the gas can normally pass through the gap. When liquid water molecules flow into the inlet of the exhaust check valve from the overflow guide hole and move towards the exhaust branch channel, the membrane suspension end is jacked upwards to block the exhaust guide hole, the channel is sealed, and the liquid water molecules are prevented from passing.
As shown in fig. 4, the upper membrane 10 is provided with a first sample adding hole 11, a first exhaust hole 12, a plurality of sample inlet sub-channels 13 arranged in parallel, a plurality of overflow channels 14 arranged in parallel, and a plurality of exhaust sub-channels 15 arranged in parallel.
As shown in fig. 3, the middle membrane includes a second sample application hole 21, a second vent hole 22, a sample application one-way valve 23, a sample application guide hole 24, a first overflow guide hole 25, a second overflow guide hole 26, an exhaust guide hole 27, and a gas-permeable water-blocking filter element 28.
The sample inlet hole of the sample inlet chamber consists of a first sample inlet hole 11 and a second sample inlet hole 21;
the exhaust hole of the sample feeding chamber consists of a first exhaust hole 12 and a second exhaust hole 22;
the first overflow guide hole 25 is an overflow guide liquid inlet hole of the overflow channel;
the second overflow guide hole 26 is an overflow guide liquid outlet hole of the overflow channel;
the number of the sample feeding sub-flow channel, the overflow flow channel, the exhaust sub-flow channel, the sample adding one-way valve, the sample adding guide hole, the overflow guide hole I, the overflow guide hole II, the exhaust guide hole and the air-permeable water-blocking filter element is equal to that of the reaction tanks and corresponds to that of the reaction tanks. That is, each reaction tank comprises a sample feeding branch channel, an overflow channel, an exhaust branch channel, a sample feeding check valve, a sample feeding guide hole, an overflow guide hole I, an overflow guide hole II, an exhaust guide hole and a breathable water-blocking filter element on the longitudinal plane where each reaction tank is located.
The first sample adding hole is vertically communicated with the second sample adding hole, and the bottom of the second sample adding hole is communicated with the first sample adding main flow channel.
And plugs made of elastic materials are preferably arranged in the first sample adding hole and the second sample adding hole, so that sample adding leakage is prevented.
The bottom inlet end of each sample adding one-way valve is communicated with the sample adding main channel, and the top outlet end of each sample adding one-way valve is communicated with the inlet of the corresponding sample adding sub-channel.
The sample adding check valve structure mainly comprises elastic materials such as silica gel and rubber, and the setting of the sample adding check valve can balance the resistance among sample feeding sub-channels, so that the sample adding hole sample is uniformly distributed to the sample feeding sub-channels.
The outlet of each sample feeding sub-channel is connected with the top end of each sample feeding guide hole, and the bottom end of each sample feeding guide hole points to the corresponding reaction tank.
The bottom end of each overflow guide hole I points to the corresponding reaction tank, and the top end of each overflow guide hole I is communicated with the inlet of the corresponding overflow channel.
The top end of each overflow guide hole II is connected with the outlet of the corresponding overflow channel, and the bottom end of each overflow guide hole II is communicated with the inlet of the corresponding exhaust check valve.
The outlet of each exhaust one-way valve is connected with the inlet of the exhaust branch channel through the corresponding exhaust guide hole, the outlet of each exhaust branch channel is communicated with the inlet of the corresponding air-permeable water-blocking filter element, and the outlet of each air-permeable water-blocking filter element is communicated with the first exhaust main channel.
In other words, in the microfluidic chip kit of the invention, the outlet of each sample injection sub-channel is communicated with the corresponding reaction tank through the corresponding sample injection guide hole;
the inlet of each overflow channel is communicated with the corresponding reaction tank through the corresponding overflow guide liquid inlet hole, and the outlet of each overflow channel is communicated with the liquid inlet of the corresponding exhaust one-way valve through the corresponding overflow guide liquid outlet hole;
the outlet of each exhaust one-way valve is connected with the inlet of an exhaust branch channel through a corresponding exhaust guide hole, the outlet of each exhaust branch channel is communicated with the inlet of a corresponding air-permeable water-blocking filter element, and the outlet of each air-permeable water-blocking filter element is communicated with an exhaust gathering port corresponding to an exhaust main channel;
the exhaust one-way valves are arranged on the bottom-layer membrane and are positioned on the inner side of the vertical section flow channel of the exhaust main flow channel, and meanwhile, the exhaust one-way valves are symmetrically distributed on two sides of the transverse straight section flow channel of the exhaust main flow channel;
the sample adding one-way valves are arranged on the middle layer chip, and the positions of the sample adding one-way valves on the middle layer chip correspond to the positions of the various branch outlets of the sample adding main flow channel one by one;
the sample adding guide hole, the overflow guide liquid inlet hole, the overflow guide liquid outlet hole and the exhaust guide hole are respectively vertically arranged on the middle layer chip in a penetrating way;
the breathable water-blocking filter cores are arranged on the middle chip, and the positions of the breathable water-blocking filter cores on the middle chip are in one-to-one correspondence with the positions of the exhaust gathering ports of the exhaust main flow channel.
The material of the air-permeable water-blocking filter element is preferably special material with a three-dimensional porous structure, such as glass fiber, polyethylene and the like, and is further preferably a 20um filter element of a Biocomma model F10-14-16.
And an outlet of the exhaust main flow channel II is communicated with the bottom of the exhaust hole II, and the exhaust hole II is vertically communicated with the exhaust hole I.
Furthermore, the sample introduction sub-runner and the overflow runner are both hot-melting structure runners. The hot melt structure flow channel is a main structure for sealing a plurality of reaction cavities of the chip, replaces a plurality of complex valve structures, and provides a quick sealing mode for the chip with a plurality of reaction structures.
During the test, the extracted nucleic acid sample enters the chip from the first sample adding hole, flows through the first sample adding main flow channel and the second sample adding main flow channel on the bottom membrane, passes through the sample adding one-way valve and enters the reaction cavity through the sample adding sub-flow channel, and the gas in the chip is discharged through the exhaust one-way valve and the air-permeable water-blocking filter element through the exhaust hole.
The different reaction cavities can be pre-fixed with reagent dry powders such as primers, fluorescent probes, enzymes and the like for detecting different target genes, and nucleic acid is mixed with the reagents after entering the reaction cavities to form reaction liquid for PCR amplification reaction.
When all the reaction cavities are filled with liquid and redundant liquid is blocked at the position of the exhaust check valve and cannot flow continuously, a heat sealing blade is used for melting and blocking a sample introduction flow channel, an overflow flow channel and the like on the upper membrane by an instrument to completely isolate all the reaction cavities from other parts, and then the chip is moved between heating modules with different temperatures, such as 95 ℃ and 60 ℃, according to a reaction program to carry out PCR amplification reaction. And after the amplification is finished, carrying out fluorescence quantitative or qualitative analysis on the reaction liquid in the reaction cavity, and determining the detection result.
The method of using the microfluidic chip kit according to the present invention is described in more detail below with reference to examples.
The primers and the probes are prepared into dry powder by drying methods such as low-temperature drying, vacuum pumping, freeze drying and the like, and are respectively fixed in each reaction hole.
Furthermore, the dry powder requires a specific reagent to dissolve the primer probe, and the reagent contains mannitol, trehalose, bovine serum albumin, tween-20 and other components.
Further, the fixing method can adopt heating to melt paraffin into liquid, dripping or spraying the liquid on the surface of the primer probe dry powder, cooling and solidifying to ensure that the dry powder is fixed in the reaction hole.
Example 1
The kit for detecting the micro-fluidic chip of 10 respiratory tract infection pathogens comprises a primer probe mixed solution, an RT-PCR buffer, a mixed enzyme solution, a positive quality control product and RNase-free water; RT-PCR buffer includes PCR buffer, dATP, dUTP, dCTP, dGTP and MgCl2(ii) a The mixed enzyme solution comprises HotStart Taq enzyme, reverse transcriptase and UNG enzyme; the positive quality control product contains plasmid corresponding to the amplification gene sequence of the 10 respiratory tract pathogens.
The final concentration of the primer in the amplification system is 100-1000 nM; the final concentration of the probe in the amplification system is 50-500 nM.
Operation and result determination of reagent kit
(1) 50 mu L of sample DNA/RNA co-extraction template (extracted from human sputum, throat swab and the like), 500 mu L of sample DNA/RNA co-extraction template is diluted by RNase-free water and added into a sample hole of a chip, the sample DNA/RNA co-extraction template is mixed with mixed enzyme liquid and RT-PCR buffer pre-filled in the sample hole, then the micro-fluidic chip is placed into a chip detector, the mixed liquid flows into 10 reaction holes respectively pre-filled with primer probes (1-10) through a flow channel under pressure, and PCR reaction and detection are started.
(2) The conditions of the PCR amplification reaction are as follows: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10 s; annealing at 60 ℃ for 35 s; 40 cycles.
(3) And (3) judging the effectiveness:
the negative control hole is a blank hole without a primer probe, the detection result is negative, the positive control hole is detected to be positive, otherwise, the experiment is considered to be invalid;
(4) and (4) interpretation of results:
the number 1-10 holes are respectively corresponding to 10 respiratory tract infection pathogens, and the machine automatically reads the fluorescence intensity value of each detection hole to judge the negative and positive results.
Example 2
And (3) detecting 15 samples by using a microfluidic chip, wherein 5 negative samples are numbered from 1 to 5, and 10 positive samples respectively containing 10 pathogen positive strain nucleic acids are numbered from 6 to 15. Sample No.6 contains mycoplasma pneumoniae nucleic acid, sample No.7 contains chlamydia pneumoniae nucleic acid, sample No. 8 contains legionella pneumophila nucleic acid, sample No.9 contains bordetella pertussis nucleic acid, sample No.10 contains rhinovirus nucleic acid, sample No. 11 contains respiratory adenovirus nucleic acid, sample No.12 contains influenza a nucleic acid, sample No.13 contains influenza b nucleic acid, sample No. 14 contains respiratory syncytial virus nucleic acid, and sample No.15 contains parainfluenza virus nucleic acid. The same procedures as in example 1 were carried out, and the results are shown in Table 1.
TABLE 1
The detection result shows that the kit has good detection specificity when being used for a microfluidic chip, only detects the corresponding pathogen, and does not generate cross reaction on nucleic acid of other pathogens.
Example 3
Respectively detecting ten pathogen plasmid mixed samples by using a microfluidic chip according to the dilution concentration of 105copies/μL、104copies/μL、103copies/μL、102Sensitivity tests were performed on five gradients, copies/. mu.L, 10 copies/. mu.L. Detection was carried out in the same manner as in example 1.
The detection result shows that the kit has good sensitivity in the microfluidic chip, and the Ct value changes in a gradient manner along with the reduction of the concentration, and the result is shown in table 2.
TABLE 2
Test results show that the kit has high sensitivity for the diagnosis of respiratory pathogens such as mycoplasma pneumoniae, chlamydia pneumoniae, legionella pneumophila, bordetella pertussis, rhinovirus, influenza A virus, influenza B virus, respiratory adenovirus, syncytial virus and parainfluenza virus in a microfluidic chip, and the detection sensitivity of each item can reach 100 copies/mu L.
Sequence listing
<110> Nanjing langyu Biotechnology Co., Ltd
<120> microfluidic chip kit for detecting 10 respiratory tract infection pathogens and using method thereof
<150>2017106650261
<151>2017-08-07
<160>30
<170>SIPOSequenceListing 1.0
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gtggtgaagt gatgcatct 19
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<213> Chlamydia pneumoniae (Chlamydophila pneumoniae artificial sequence)
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<213> Chlamydia pneumoniae (Chlamydophila pneumoniae artificial sequence)
<400>6
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<213> Legionella pneumophila (Legionella pneumophila artificial sequence)
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<400>8
ccgatatctg tgccatatc 19
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<213> Legionella pneumophila (Legionella pneumophila artificial sequence)
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<213> respiratory Adenovirus (Adenovirus artificial sequence)
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ccatctcctt cgataaag 18
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<212>DNA
<213> Influenza A Virus (Influenza A Virus Artificial sequence)
<400>19
gaagcccgtt gaagacta 18
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<212>DNA
<213> Influenza A Virus (Influenza A Virus Artificial sequence)
<400>20
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<212>DNA
<213> Influenza A Virus (Influenza A Virus Artificial sequence)
<400>21
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gagatggagt caggtggtt 19
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<213> Influenza B virus (Influenza B virus artificial sequence)
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<213> Influenza B virus (Influenza B virus artificial sequence)
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<213> respiratory syncytial virus (Artificial sequence of respiratory syncytial virus)
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<213> parainfluenza Virus (human parainfluenza Virus Artificial sequence)
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Claims (8)
1. A micro-fluidic chip kit for detecting 10 respiratory tract infection pathogens comprises a kit body, wherein the kit body comprises a sample introduction chamber, a reaction chamber and a micro-fluidic flow channel for realizing the communication of the sample introduction chamber and the reaction chamber; the method is characterized in that: the reaction chambers at least comprise 10 reaction chambers, each reaction chamber is mutually independent, and each reaction chamber is pre-provided with reagent dry powder for amplifying a respiratory tract pathogen, and the reagent dry powder comprises a primer and a TaqMan probe for amplifying the corresponding respiratory tract pathogen;
the reagent dry powder preset in each reaction chamber can amplify any one of the following 10 respiratory pathogens, and meanwhile, the respiratory pathogens amplified by the reagent dry powder in all the reaction chambers can include the following 10 respiratory pathogens: mycoplasma pneumoniae (Mycoplasma pneumoniae), chlamydia pneumoniae (chlamydophila), Legionella pneumophila (Legionella pneumophila), bordetella pertussis (bordethouse), rhinoviruses (Human rhinoviruses), respiratory adenoviruses (Adenovirus), Influenza a viruses (inflenza a viruses), Influenza B viruses (Influenza B viruses), respiratory syncytial viruses (respiratory syncytial viruses), and parainfluenza viruses (Human parainfluenza viruses);
the sample introduction cavity is arranged at the middle position of the kit body and is provided with a sample introduction hole, a sample outlet, an air inlet hole and an air exhaust hole;
the micro-fluidic runner includes and advances kind sprue, advances kind subchannel, exhaust sprue, exhaust subchannel and overflow channel, wherein: the number of the sample feeding branch channels, the exhaust branch channels and the overflow channels is consistent with that of the reaction chambers;
the sample outlet of the sample injection cavity is communicated with one end of a sample injection main flow channel, the other end of the sample injection main flow channel is provided with sample branch flow outlets the number of which is consistent with that of the reaction cavities, and the sample branch flow outlets of the sample injection main flow channel are communicated with the sample injection ports of the corresponding reaction cavities one by one through sample injection branch flow channels; each sample feeding branch channel is provided with a sample feeding one-way valve;
the air inlet of the sample introduction cavity is communicated with one end of the main exhaust channel, the other end of the main exhaust channel is provided with exhaust gathering ports with the number consistent with that of the reaction cavities, and each exhaust gathering port of the main exhaust channel is communicated with the air inlet of the corresponding reaction cavity one by one through an exhaust branch channel, an exhaust check valve, a breathable water-blocking filter element and an overflow channel in sequence;
the sequence of the primer is as follows: mycoplasma pneumoniae (Mycoplasma pneumoniae), SEQ ID nos. 1-2; chlamydia pneumoniae (Chlamydophila pneumoniae), SEQ ID nos. 4 to 5; legionella pneumophila (legionella pneumophila), SEQ ID No. 7-8; bordetella pertussis (Bordetella pertussis), SEQ ID No. 10-11;
rhinoviruses (Human rhinoviruses), SEQ ID NO. 13-14; respiratory Adenovirus (Adenovirus), SEQ ID Nos. 16-17; influenza a virus (Influenza a virus), SEQ ID No. 19-20; influenza B virus (Influenza B virus), SEQ ID NO. 22-23; respiratory syncytial virus (respiratory syncytial virus), SEQ ID Nos. 25 to 26; parainfluenza virus (human parainfluenza virus), SEQ ID Nos. 28 to 29;
the sequence of the TaqMan probe is as follows: mycoplasma pneumoniae (Mycoplasma pneumoniae), SEQ ID No. 3; chlamydia pneumoniae (Chlamydophila pneumoniae), SEQ ID No. 6; legionella pneumophila (legionella pneumophila), SEQ ID No. 9; bordetella pertussis (Bordetella pertussis), SEQ ID No. 12; rhinovirus (Human rhinovirus), SEQ ID No. 15; respiratory Adenovirus (Adenovirus), SEQ ID No. 18; influenza a virus (Influenza a virus), SEQ ID No. 21; influenza B virus (Influenza B virus), SEQ id No. 24; respiratory syncytial virus (respiratory syncytial virus), SEQ ID No. 27; parainfluenza virus (human parainfluenza virus), SEQ ID NO. 30.
2. The microfluidic chip kit for detecting 10 respiratory tract infection pathogens according to claim 1, wherein the kit comprises: the final concentration of the primer in the amplification system is 100-1000 nM; the final concentration of the TaqMan probe in an amplification system is 50-500 nM.
3. The microfluidic chip kit for detecting 10 respiratory tract infection pathogens according to claim 1, wherein the kit comprises: an RT-PCR buffer, a mixed enzyme solution and a positive quality control product are preset in the sample introduction chamber.
4. The microfluidic chip kit for detecting 10 respiratory tract infection pathogens according to claim 3, wherein the kit comprises: the RT-PCR buffer comprises PCR buffer, dATP, dUTP, dCTP, dGTP and MgCl2。
5. The microfluidic chip kit for detecting 10 respiratory tract infection pathogens according to claim 3, wherein the kit comprises: the mixed enzyme solution comprises HotStart Taq enzyme, reverse transcriptase and UNG enzyme.
6. The microfluidic chip kit for detecting 10 respiratory tract infection pathogens according to claim 3, wherein the kit comprises: the positive quality control product contains plasmids corresponding to the 10 respiratory tract pathogen amplification gene sequences.
7. The microfluidic chip kit for detecting 10 respiratory tract infection pathogens according to claim 1, wherein the kit comprises: the kit body is of a three-piece structure and comprises an upper-layer membrane, a middle-layer membrane and a bottom-layer membrane which are hermetically bonded from top to bottom in sequence; wherein: the sample introduction cavity comprises a sample introduction groove, and a sample introduction hole and an exhaust hole of the sample introduction cavity are through holes which penetrate through the upper layer membrane and the middle layer membrane and are communicated with the reaction groove; the reaction chamber comprises a reaction tank;
the sample injection main flow passage and the exhaust main flow passage are respectively communicated with the sample injection groove, the sample injection main flow passage and the exhaust main flow passage are all arranged on the bottom layer membrane, and the sample injection main flow passage and the exhaust main flow passage are both T-shaped flow passages and are respectively arranged at two sides of the sample injection groove;
the T-shaped flow channel comprises a transverse straight section flow channel and a vertical section flow channel, the transverse straight section flow channel is arranged in the middle of the bottom membrane and is communicated with the sample feeding groove, and the vertical section flow channel is arranged along the side edge of the bottom membrane;
the various branch flow outlets of the sample adding main flow channel are arranged on the vertical section flow channel of the sample adding main flow channel, and the exhaust convergence ports of the exhaust main flow channel are arranged on the vertical section flow channel of the exhaust main flow channel;
the reaction tanks are arranged on the bottom-layer diaphragm and are positioned on the inner side of the vertical section runner of the main sampling runner, and meanwhile, the reaction tanks are symmetrically distributed on two sides of the horizontal straight section runner of the main sampling runner;
the sample injection sub-channel, the exhaust sub-channel and the overflow channel are respectively arranged on the middle layer chip, wherein:
the outlet of each sample feeding branch channel is communicated with the corresponding reaction tank through a corresponding sample feeding guide hole;
the inlet of each overflow channel is communicated with the corresponding reaction tank through the corresponding overflow guide liquid inlet hole, and the outlet of each overflow channel is communicated with the liquid inlet of the corresponding exhaust one-way valve through the corresponding overflow guide liquid outlet hole;
the outlet of each exhaust one-way valve is connected with the inlet of an exhaust branch channel through a corresponding exhaust guide hole, the outlet of each exhaust branch channel is communicated with the inlet of a corresponding air-permeable water-blocking filter element, and the outlet of each air-permeable water-blocking filter element is communicated with an exhaust gathering port corresponding to an exhaust main channel;
the exhaust one-way valves are arranged on the bottom-layer membrane and are positioned on the inner side of the vertical section flow channel of the exhaust main flow channel, and meanwhile, the exhaust one-way valves are symmetrically distributed on two sides of the transverse straight section flow channel of the exhaust main flow channel;
the sample adding one-way valves are arranged on the middle layer chip, and the positions of the sample adding one-way valves on the middle layer chip correspond to the positions of the various branch outlets of the sample adding main flow channel one by one;
the sample adding guide hole, the overflow guide liquid inlet hole, the overflow guide liquid outlet hole and the exhaust guide hole are respectively vertically arranged on the middle layer chip in a penetrating way;
the breathable water-blocking filter cores are arranged on the middle chip, and the positions of the breathable water-blocking filter cores on the middle chip are in one-to-one correspondence with the positions of the exhaust gathering ports of the exhaust main flow channel.
8. The microfluidic chip kit for detecting 10 respiratory tract infection pathogens according to claim 7, wherein the kit comprises: the exhaust check valve comprises a diaphragm, one end of the diaphragm is pressed between the middle diaphragm and the bottom diaphragm, the other end of the diaphragm is suspended, and a diaphragm caulking groove is formed in the bottom diaphragm at a position corresponding to the diaphragm suspension end; the diaphragm suspension end is opposite to the exhaust guide hole, the size of the outer edge of the diaphragm suspension end is larger than the aperture of the exhaust guide hole and smaller than the size of the inner edge of the diaphragm caulking groove, and meanwhile, a gap which only allows gas to pass through is formed between the outer edge of the diaphragm suspension end and the inner wall of the diaphragm caulking groove.
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