CN115851423A

CN115851423A - Micro-fluidic chip for detecting respiratory tract pathogen nucleic acid and detection method

Info

Publication number: CN115851423A
Application number: CN202211279873.1A
Authority: CN
Inventors: 阎旭; 盛以龙
Original assignee: Tianjin Zhishan Biotechnology Co ltd
Current assignee: Tianjin Zhishan Biotechnology Co ltd
Priority date: 2022-10-19
Filing date: 2022-10-19
Publication date: 2023-03-28

Abstract

The invention relates to a micro-fluidic chip for detecting nucleic acid of respiratory pathogens, which comprises a plurality of independent chambers, wherein the number of the chambers is more than or equal to 3; at least 1 chamber coated for GAPDH detection of upstream and downstream primers and probes and at least 1 chamber coated for Bacillus atrophaeus DNA upstream and downstream primers and probes; the other chambers are coated with primer sets and probes for detecting one or more of the following pathogen genes: novel coronavirus N gene, novel coronavirus 1ab gene, coronavirus 229E type, coronavirus OC43 type, coronavirus NL63 type, coronavirus HKU1 type, adenovirus general purpose type, respiratory syncytial virus A type and B type, human metapneumovirus, rhinovirus, influenza A type, influenza B type, human parainfluenza virus 1 type, human parainfluenza virus 2 type, human parainfluenza virus 3 type, human parainfluenza virus 4 type, and mycoplasma pneumoniae. The invention can realize the synchronous detection of various respiratory pathogens, has simple, convenient and quick operation and reduces the pollution probability.

Description

Micro-fluidic chip for detecting respiratory tract pathogen nucleic acid and detection method

Technical Field

The invention belongs to the technical field of nucleic acid amplification, and particularly relates to a micro-fluidic chip for detecting respiratory tract pathogen nucleic acid and a detection method.

Background

The common diseases of acute respiratory tract infection include acute upper respiratory tract infection, acute tracheo-bronchopneumonia, bronchiectasis and the like, and the clinical manifestations are inconsistent. Pathogens causing respiratory tract infection are various in types, such as viruses, bacteria, mycoplasma, chlamydia, legionella and other microorganisms; and one pathogen may cause multiple clinical manifestations, which in turn may be caused by multiple pathogens. In clinic, pathogen is difficult to treat, the treatment is not symptomatic, some patients have long-term fever and even become worse, or antibiotics are abused. Therefore, the multiple detection of pathogens of respiratory tract infection has important significance for clinical timely diagnosis and epidemic situation report.

About 70% -80% of acute upper respiratory infections are caused by viruses. Mainly comprises influenza virus (A, B and C), parainfluenza virus, respiratory syncytial virus, adenovirus, rhinovirus, coxsackie virus, measles virus and rubella virus. Bacterial infections can occur directly or following viral infections, with hemolytic streptococci being common, followed by haemophilus influenzae, pneumococcus and staphylococci, among others. Gram-negative bacilli are occasionally observed. The main manifestations of the infection are rhinitis, pharyngolaryngitis or tonsillitis. 80% of the upper respiratory tract diseases and some of the lower respiratory tract diseases are caused by pathogens other than bacteria. Among them, atypical pathogens including respiratory viruses are the most common, such as legionella pneumophila, mycoplasma pneumoniae, chlamydia pneumoniae, adenovirus, respiratory syncytial virus, influenza virus, parainfluenza virus and other microorganisms, and the etiology is not only complex but often mixed. Accurate etiology analysis is not only a basis for diagnosis, but also a basis for rational selection of treatment regimens.

Currently, common detection methods for detecting respiratory pathogens include: isolation and culture of pathogens and tissue cell culture methods, serology, direct detection methods (including electron microscopy), indirect and direct immunofluorescence antibody methods (IFA/DFA), enzyme immunoassay, nucleic acid amplification methods, and the like. The isolation culture and tissue cell culture methods of pathogens are often used as gold standards, but have the defects of complex operation, long culture time, great technical difficulty, low positive rate and the like. Although the electron microscope detection method is used for directly detecting pathogen particles, the detection positive rate is not high, the detection time is long, and the method is not suitable for clinical rapid diagnosis. The nucleic acid detection is performed by hybridization or polymerase chain reaction technology, has strong sensitivity and high specificity, and can perform trace detection. However, hybridization PCR is performed after amplification, and an operation of taking out PCR products by opening a tube is required, so that high-concentration products are exposed to the air, and cross contamination among samples is easily caused.

Therefore, the development of a respiratory tract pathogen detection scheme which can simultaneously detect multiple pathogens, is simple, convenient and quick to operate and can effectively prevent cross contamination has important significance for solving the problems in the prior art.

Disclosure of Invention

Technical problem to be solved

In view of the above disadvantages and shortcomings of the prior art, the present invention provides a microfluidic chip for detecting nucleic acid of respiratory pathogens, which can detect multiple pathogens at the same time, and has the advantages of simple and rapid operation, and closed system amplification to effectively prevent cross contamination. The invention also relates to a method for detecting respiratory tract pathogens by using the microfluidic chip.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

in a first aspect, the present invention provides a microfluidic chip for detecting nucleic acid of a respiratory pathogen, which comprises a plurality of independent chambers, wherein each chamber is respectively communicated with a sample injection hole and an exhaust hole; the number of the chambers is more than or equal to 3;

at least 1 chamber of the plurality of chambers is internally coated with upstream and downstream primers and probes for detecting GAPDH, and at least 1 chamber is internally coated with upstream and downstream primers and probes for detecting Bacillus atrophaeus DNA;

the other chambers are each independently coated with upstream and downstream primers and probes for detecting one or more of the following pathogen genes: novel coronavirus N gene, novel coronavirus 1ab gene, coronavirus 229E type, coronavirus OC43 type, coronavirus NL63 type, coronavirus HKU1 type, adenovirus general purpose type, respiratory syncytial virus A type, respiratory syncytial virus B type, human metapneumovirus, rhinovirus, influenza A type, influenza B type, human parainfluenza virus 1 type, human parainfluenza virus 2 type, human parainfluenza virus 3 type, human parainfluenza virus 4 type, and mycoplasma pneumoniae.

According to a preferred embodiment of the present invention, the upstream and downstream primers for detecting GAPDH are set forth in SEQ ID NO:55 and SEQ ID NO:56, the probe is shown as SEQ ID NO: shown as 57; the upstream and downstream primers for detecting Bacillus atrophaeus DNA are shown as SEQ ID NO:58 and SEQ ID NO:59, and the probe is shown as SEQ ID NO: as shown at 60.

According to the preferred embodiment of the present invention, the upstream and downstream primers for detecting influenza A virus are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the probe is shown in SEQ ID NO. 3; the upstream and downstream primers for detecting the influenza B virus are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the probe is shown as SEQ ID NO. 6;

the upstream and downstream primers for detecting the human parainfluenza virus type 1 are shown as SEQ ID NO. 7 and SEQ ID NO. 8, and the probe is shown as SEQ ID NO. 9;

the upstream and downstream primers for detecting the human parainfluenza virus type 2 are shown as SEQ ID NO 10 and SEQ ID NO 11, and the probe is shown as SEQ ID NO 12;

upstream and downstream primers for detecting the human parainfluenza virus type 3 are shown as SEQ ID NO 13 and SEQ ID NO 14, and a probe is shown as SEQ ID NO 15;

the upstream and downstream primers for detecting the human parainfluenza virus type 4 are shown as SEQ ID NO 16 and SEQ ID NO 17, and the probe is shown as SEQ ID NO 18;

the upstream and downstream primers for detecting the adenovirus universal type are shown as SEQ ID NO. 19 and SEQ ID NO. 20, and the probe is shown as SEQ ID NO. 21;

the upstream and downstream primers for detecting coronavirus 229E are shown as SEQ ID NO. 22 and SEQ ID NO. 23, and the probe is shown as SEQ ID NO. 24;

the upstream and downstream primers for detecting coronavirus NL63 are shown as SEQ ID NO. 25 and SEQ ID NO. 26, and the probe is shown as SEQ ID NO. 27;

the upstream and downstream primers for detecting coronavirus HKU1 type are shown as SEQ ID NO 28 and SEQ ID NO 29, and the probe is shown as SEQ ID NO 30;

the upstream and downstream primers for coronavirus OC43 type are shown as SEQ ID NO. 31 and SEQ ID NO. 32, and the probe is shown as SEQ ID NO. 33;

upstream and downstream primers for detecting respiratory syncytial virus type A are shown as SEQ ID NO. 34 and SEQ ID NO. 35, and a probe is shown as SEQ ID NO. 36;

upstream and downstream primers for detecting respiratory syncytial virus type B are shown as SEQ ID NO. 37 and SEQ ID NO. 38, and a probe is shown as SEQ ID NO. 39;

the upstream and downstream primers for detecting Mycoplasma pneumoniae are shown as SEQ ID NO 40 and SEQ ID NO 41, and the probe is shown as SEQ ID NO 42;

the upstream and downstream primers for detecting the human metapneumovirus are shown as SEQ ID NO 43 and SEQ ID NO 44, and the probe is shown as SEQ ID NO 45;

the upstream and downstream primers for detecting rhinovirus are shown as SEQ ID NO 46 and SEQ ID NO 47, and the probe is shown as SEQ ID NO 48;

upstream and downstream primers for detecting the N gene of the novel coronavirus are shown as SEQ ID NO. 49 and SEQ ID NO. 50, and a probe is shown as SEQ ID NO. 51;

the upstream and downstream primers for detecting the novel coronavirus 1ab gene are shown as SEQ ID NO. 52 and SEQ ID NO. 53, and the probe is shown as SEQ ID NO. 54.

According to a preferred embodiment of the present invention, at least 1 of the plurality of chambers is empty and not coated with any detection reagent.

According to the preferred embodiment of the invention, in the process of coating the primers and the probes in each chamber, a 1uL primer group and probe mixture is taken by a liquid transfer machine and spotted into the corresponding chamber, air-dried and then the coating film of the chamber is sealed; wherein, in the mixture of the primer group and the probe, the total concentration of the primer group and the probe is 0.4-1 mu mol/L, preferably 0.5 mu mol/L.

According to the preferred embodiment of the present invention, the number of chambers is 20 or more.

According to a preferred embodiment of the invention, the microfluidic chip comprises a detection sheet body, wherein the detection sheet body is a sheet-shaped plate body and comprises a first side surface and a second side surface, a plurality of cavity holes, branch sample injection flow channels communicated with first ends of the cavity holes, exhaust flow channels communicated with second ends, exhaust holes communicated with the exhaust flow channels, a main flow channel communicated with the branch sample injection flow channels and sample injection holes positioned at the starting end of the main flow channel are formed in the first side surface in a concave mode, and the cavity holes are of a through hole structure; the branch sample injection flow passage, the exhaust flow passage and the exhaust hole are of non-through hole structures;

the opening side of the exhaust hole is covered with a hydrophobic breathable film, and a first sealing sheet is compounded on the first side surface of the detection sheet body to seal the branch sample injection flow channel, the exhaust flow channel and the first side of the cavity hole; the second side surface of the detection sheet body is compounded with a second sealing sheet so as to seal the second side of the cavity hole, so that the cavity hole becomes a sealed cavity;

the micro-fluidic chip also comprises a third sealing sheet and a cover plate which are used for sealing the exhaust holes and the sample injection holes after sample injection is finished.

According to the preferred embodiment of the invention, the horizontal section of the chamber is crescent, oval or spindle-shaped, the width of each chamber is gradually reduced from the middle to the two ends, and the chamber is provided with a convex cambered surface; the convex cambered surface is opposite to the peripheral edge facing the detection sheet body. When the cavity with the structure is used for detecting probe signals in the cavity by adopting an optical instrument from the peripheral edge, the convex cambered surface has the light-gathering effect of the convex lens, so that higher optical signals can be obtained.

According to a preferred embodiment of the invention, the chamber has an aspect ratio of between 3:1.4-2, length-thickness ratio between 3. Preferably, the chamber has a length of 2.95-3.1mm, a width of 1.5-1.7mm and a thickness (depth) of 1.2-1.4mm.

According to a preferred embodiment of the invention, the volume of the chamber is 5-10 μ L.

In a second aspect, the present invention provides a method for detecting nucleic acid of respiratory tract pathogen, wherein the method uses the above microfluidic chip to perform nucleic acid detection, and the detection steps are as follows:

s1, sample processing and template extraction are carried out to obtain a template to be detected;

s2, preparing an RT-PCR amplification reagent, and mixing the RT-PCR amplification reagent with a template to be detected to prepare a liquid amplification system reagent;

s3, injecting a liquid amplification system reagent from the sample injection hole of the microfluidic chip to fill each chamber with the liquid amplification system reagent, and then sealing the sample injection hole and the exhaust hole;

s4, placing the microfluidic chip into a nucleic acid amplification instrument for temperature-variable controlled amplification;

and S5, performing optical detection on each chamber by using a nucleic acid amplification instrument, and performing data processing and analysis.

(III) advantageous effects

The invention designs a series of primers and probes for detecting common nucleic acids of various respiratory pathogens, sets a primer group and a probe for detecting an extraction control (GAPDH gene) and an amplification control (Bacillus atrophaeus DNA), and coats the primers and the probe in each independent and closed chamber of a microfluidic chip in advance respectively, and can realize synchronous detection and analysis of various pathogens only by once sampling and pretreatment (preparation of a liquid reagent of an RT-PCR amplification system).

The micro-fluidic chip provided by the invention can detect a plurality of types of pathogens at one time, and has the advantages of high sensitivity, strong specificity and the like; meanwhile, the micro-fluidic chip provided by the invention can monitor the amplification condition in real time through a fluorescent amplification curve, avoids the process of hybridization after amplification of the traditional gene chip, is simple and convenient to operate and quick to detect, and reduces the possibility of pollution because the whole amplification and detection process is in a closed state, and the cost is low and quick.

Drawings

Fig. 1 is a schematic structural diagram of a microfluidic chip according to the present invention.

FIGS. 2 to 20 show the amplification curves of primer IFA-for circulation A, primer IFB-for amplification curve for circulation B, primer PIV 1-for amplification curve for parainfluenza, PIV 2-for amplification curve for parainfluenza, PIV 3-for amplification curve for parainfluenza, PIV 4-for amplification curve for parainfluenza, OC 43-for amplification curve for coronavirus, HKU 1-for amplification curve for coronavirus, 229E-for amplification curve for coronavirus, NL 63-for amplification curve for coronavirus, MP-for amplification curve for mycoplasma pneumoniae, RSV-for amplification curve for syncytial virus, ADV-for adenovirus, HMPV-for metapneumovirus, RV-for rhinovirus, 2019-nCOV-1-for amplification curve for new coronavirus, N2019-nCOV-1-for new coronavirus, GAGAGAP (EC) -for amplification curve, and Bacillus subtilis amplification curve for quality control.

Fig. 21 is an exploded view of a microfluidic chip according to a preferred embodiment of the present invention.

Fig. 22 is a schematic overall structure diagram of a microfluidic chip according to a preferred embodiment of the present invention.

Fig. 23 is a schematic diagram of a chamber structure of a microfluidic chip according to a preferred embodiment of the present invention.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings. In the following examples, unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the experimental procedures are conventional procedures well known to those skilled in the art.

The invention mainly provides a micro-fluidic chip technology and a detection method capable of simultaneously detecting multiple respiratory pathogens, which are not limited to be applied on a micro-fluidic chip with a specific structure, and the basic characteristics of the micro-fluidic chip integrated with a plurality of independent chambers meet the requirements of the invention as long as the basic characteristics can meet the requirements: the microfluidic chip comprises a plurality of independent chambers, wherein each chamber is respectively communicated with a sample injection hole and an exhaust hole; the number of the chambers is more than or equal to 3.

Of these chambers, at least 1 chamber is internally coated with upstream and downstream primers and probes for detecting GAPDH, and at least 1 chamber is internally coated with upstream and downstream primers and probes for detecting bacillus atrophaeus DNA; the other chambers are each independently coated with upstream and downstream primers and probes for detecting one or more of the following pathogen genes: novel coronavirus N gene, novel coronavirus 1ab gene, coronavirus 229E type, coronavirus OC43 type, coronavirus NL63 type, coronavirus HKU1 type, adenovirus general purpose type, respiratory syncytial virus A type, respiratory syncytial virus B type, human metapneumovirus, rhinovirus, influenza A type, influenza B type, human parainfluenza virus 1 type, human parainfluenza virus 2 type, human parainfluenza virus 3 type, human parainfluenza virus 4 type, and mycoplasma pneumoniae.

The following are preferred embodiments of the present invention.

Example 1

As shown in fig. 1, the microfluidic chip 1 includes a sample injection hole 11 and a plurality of independent chambers 15, one end of each chamber 15 is connected to a branch sample injection channel 132, and the other end of each chamber 15 is connected to an exhaust channel 121. Each branch sample injection flow channel 132 is directly or indirectly communicated with the sample injection hole 11 (the branch sample injection flow channel 132 is communicated with the total flow channel 131, and the total flow channel 131 is communicated with the sample injection hole 11), and the exhaust flow channel 121 is communicated with the exhaust hole 12. Wherein each chamber 15 communicates with at least one venting orifice 12. Each chamber 15 is coated with a mixture of probes of primer sets for one pathogen, respectively, to enable simultaneous detection and analysis of multiple pathogen nucleic acids. Of course, the structure of the microfluidic chip is not limited to the above. For example, a plurality of sample injection holes may be provided, and the number of sample injection holes corresponds to the number of chambers, i.e., each chamber is independently communicated with one sample injection hole through a liquid inlet flow passage and one exhaust hole through an exhaust flow passage. However, the structure is complex, and multiple sample feeding operations are required during detection, which is inconvenient for rapid detection.

The invention provides a micro-fluidic chip capable of detecting various respiratory pathogens simultaneously, which comprises the following preparation steps:

(I) selection of respiratory pathogens

The pathogen primer group and the probe coated in the microfluidic chip at least comprise the primer group and the probe for detecting the following pathogen genes: novel coronavirus N gene (SARS-Cov-2-N), novel coronavirus 1ab gene, coronavirus 229E type, coronavirus OC43 type, coronavirus NL63 type, coronavirus HKU1 type, adenovirus universal type, respiratory syncytial virus A type and B type, human metapneumovirus, rhinovirus, influenza A virus, influenza B virus, human parainfluenza virus (HPIV 1), human parainfluenza virus (HPIV 2), human parainfluenza virus (HPIV 3), human parainfluenza virus (HPIV 4) and Mycoplasma pneumoniae.

It has now been demonstrated that most respiratory diseases are caused by pathogens other than bacteria, with respiratory viruses being the most common. The clinical symptoms and physical signs caused by respiratory tract infection are similar, the clinical manifestations mainly comprise rhinitis, pharyngitis, laryngitis, tonsillitis and other symptoms, and tracheitis, bronchitis, pneumonia and the like can be caused seriously, but the treatment method, the curative effect and the course of the infection caused by different pathogens are different. In order to grasp the cause of the disease in time and treat the pathogen as early as possible, the pathogen species causing the disease needs to be rapidly detected in a plurality of similar diseases, and when the pathogen primer group and the probe with known species are not positive, the new epidemic situation is possible. Therefore, the micro-fluidic chip for detecting the respiratory tract pathogen nucleic acid has important significance for synchronously carrying out multi-detection on the respiratory tract infection pathogens, and clinically and timely diagnosing and reporting epidemic situations.

(II) design of primer set and Probe

The specific target gene markers of each pathogen are determined by consulting literature and analyzing pathogen genome sequences, and then are designed according to the parameter requirements that the annealing temperature is 60 ℃, the length of an amplification product is 100-150bp, the Tm value of a probe is at least 5 degrees higher than that of a Primer and the like by combining Primer Premier5, beacon designer software and Primer-BLAST (NCBI) analysis, and the probe sequences of the corresponding primers of each pathogen are shown in Table 1. After design, the probe is delivered to a company with synthetic qualification for synthesis, wherein the 5 'end of the probe is marked with FAM group, and the 3' end of the probe is marked with BHQ1 or MGB.

In addition, in order to monitor the quality of the sample and the amplification reagent, an extraction control and an amplification control need to be set, wherein the extraction control generally selects a housekeeping gene (also called housekeeping gene) which is stably expressed in a human body, and the GAPDH gene is selected in the invention; the amplification control selects genes with lower or no homology with the pathogen to be detected, the Bacillus atrophaeus gene is selected, and the two genes are subjected to primer probe design according to the same parameter requirements as the pathogen.

Finally, the primer groups and probe sequences pre-coated in different chambers of the microfluidic chip are determined as shown in table 1.

Table 1: pre-coated primer group and probe in microfluidic chip

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F-containing represents an upstream primer, R-containing represents a downstream primer and P-containing represents a probe in the primer names; the primer sequence of SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 19 or SEQ ID NO. 39 has Y as C or T, K as G or T, and R as A or G.

(III) preparation of primer-Probe mixture

After synthesizing the upstream and downstream primer sets and probes designed for each pathogen listed in Table 1, the primers and probes were dissolved in TE solution to prepare 10. Mu. Mol/L stock solutions, and then prepared in the proportions shown in Table 2 to prepare primer-probe mixtures with a total concentration of 1. Mu. Mol/L, which were stored at-20 ℃ for further use.

When preparing the amplification control (Bacillus atrophaeus) primer probe mixture, replace the water in Table 2 with 1X 10^5 copies/. Mu.L Bacillus atrophaeus genomic DNA.

TABLE 2 preparation of primer Probe mixtures

Components	Volume (μ L)
		Upstream primer (10. Mu. Mol/L)	0.1
Downstream primer (10. Mu. Mol/L)	0.1
		Probe (10. Mu. Mol/L)	0.1
TE	0.7
		Total of	1

(IV) coating primer probe mixtures in different chambers of the microfluidic chip

(1) Taking the micro-fluidic chip (the second side surface of the detection piece body is sealed by a membrane, and the first side surface is in an open state), ultrasonically cleaning the micro-fluidic chip in acetone for 10 minutes, then ultrasonically cleaning the micro-fluidic chip in absolute ethyl alcohol for 10 minutes, then ultrasonically cleaning the micro-fluidic chip in deionized water for 10 minutes, and finally taking the micro-fluidic chip out and drying the micro-fluidic chip in an oven for later use;

(2) And placing the microfluidic chip on a clean working table, taking 1uL primer probe mixture by using a pipette, spotting the mixture into different cavities, naturally drying the mixture, and arranging all pathogens and controls in the chip as shown in figure 1 and table 3.

(3) The hot press is used for laminating a film on the chip to form a closed microfluidic chip;

(4) And vacuumizing and packaging the microfluidic chip by using a vacuum device, and storing in a dark place for later use.

TABLE 3 detection of pathogens by chip reaction cell correspondence

Note: the lower row of chambers of the chip shown in FIG. 1 corresponds to # 1-10 from left to right in sequence, and the upper row of chambers corresponds to # 11-20 from right to left in sequence. And a primer and probe mixture for detecting the syncytial virus A and B is coated in the 3# chamber so as to ensure the detection rate.

The method for detecting the pathogens by using the micro-fluidic chip for detecting the respiratory tract pathogen nucleic acid with the structure comprises the following steps:

step 1: sample processing and template extraction

Take a throat swab as an example. 200uL of freshly collected throat swab was extracted with Viral RNA extraction Kit (CW 3127-S) produced by Jiangsukang Shidai corporation or QIAamp Viral RNA Mini Kit (52904) produced by QIAGEN corporation, following the strict instructions, and finally eluted with 50uL of nuclease-free water.

Step 2: preparation of amplification System

The RT-PCR amplification system was prepared by using One Step RT-PCR Kit (Q223-1) from Novozam. Preparing a reaction system (reaction liquid is prepared on ice), shaking and mixing uniformly, and centrifuging for a short time.

And step 3: chip loading

Sucking the amplification system by a 200 mu L liquid transfer device, injecting liquid into the microfluidic chip, pulling out the head of the liquid transfer device after the microfluidic chip is filled with the liquid, and sealing the sample adding hole by using the sealant.

And 4, step 4: PCR amplification

The microfluidic chip was placed in a nucleic acid amplification analyzer (equipment type or brand) and amplified according to the set cycle parameters shown in table 4.

TABLE 4PCR amplification cycle parameters

And 5: analysis of results

After amplification, the nucleic acid amplification analyzer carries out data processing by self-contained software, and the data is compared with the blank control hole, the detection hole judges that the detection hole has an S-shaped curve on the nucleic acid amplification analyzer equipment and is positive, and the detection hole does not see the S-shaped curve and judges that the detection hole is negative.

In order to verify the detection effect of the microfluidic chip, a clinical positive sample is collected, part of pathogens which cannot be collected into the clinical positive sample are prepared into a simulated pharyngeal swab positive sample by adopting plasmid DNA or pseudovirus, all samples are mixed and then are detected by adopting the method, and the detection result is shown in figures 2-20: FIG. 2 shows FluA as positive, FIG. 3 shows FluB as positive, FIG. 4 shows PIV1 as positive, FIG. 5 shows PIV2 as positive, FIG. 6 shows PIV3 as positive, FIG. 7 shows PIV4 as positive, FIG. 8 shows HcoV OC43 as positive, FIG. 9 shows HcoV HKU1 as positive, FIG. 10 shows HcoV 229E as positive, FIG. 11 shows HcoV NL63 as positive, FIG. 12 shows MP as positive, FIG. 13 shows RSV as positive, FIG. 14 shows AdV as positive, FIG. 15 shows HMPV as positive, FIG. 16 shows RV as positive, FIG. 17 shows 2019-nCoV-ORF 1ab as positive, FIG. 18 shows 2019-nCoV-N as positive.

Example 2

In order to improve the detection accuracy, the structure of the microfluidic chip can be designed as shown in fig. 21-22, and the coated primer group and probe mixture are all shown in example 1.

In this embodiment, the microfluidic chip includes a detection sheet body 1, and the detection sheet body 1 is provided with a plurality of cavities 11 and a plurality of vent holes 12 that are communicated in a one-to-one correspondence manner. Still be equipped with on detecting piece body 1 and annotate the appearance hole 13, cavity 11 and notes appearance hole 13 intercommunication, the first side of detecting piece body 1 is equipped with the first side of first seal membrane 2 with sealed cavity 11, the first side of exhaust hole 12 and annotates the first side of appearance hole 13, the second side of detecting piece body 1 is equipped with the second side of second seal membrane 3 with sealed cavity 11, the second side of detecting piece body 1 still is equipped with the second side of hydrophobic ventilated membrane 4 with sealed exhaust hole 12, the horizontal cross-section of cavity 11 is crescent structure. The hydrophobic breathable film 4 is a multi-layer composite material, preferably a non-water-absorbing material, so that the hydrophobic breathable film 4 is prevented from absorbing liquid from the exhaust hole 12 in the amplification reaction to cause bubbles in the chamber 11, and further the detection accuracy is influenced.

Preferably, as shown in fig. 21 and 23, the chambers 11 are not regular crescent shaped, and the length-width ratio of the single chamber 11 is between 3:1.4-2, length-thickness ratio between 3. In the embodiment shown in figure 23, the chamber 11 is 3mm in length, 1.7mm in width and 1.2mm in thickness. The chambers 11 are designed to have a long length, so that each chamber 11 has enough optical detection surface to facilitate optical detection, and the thickness needs to be as thin as possible under the condition of satisfying the molding condition of the chip injection molding process, so that the double-sided heating instrument is adapted and the heating efficiency is improved. The width of the chamber is adjusted according to the designed volume of the chamber 11, but preferably the aspect ratio is between 3:1.4-2, so that the circular arc side wall has enough curvature, which is beneficial to reducing the residual quantity of bubbles in the liquid reagent, playing the light-gathering effect of the convex mirror and enhancing the optical detection signal. The volume of the chamber 11 is 5-10 μ L (for example, 5 μ L, 6 μ L, 8 μ L or 10 μ L can be set), but the invention is not limited to the specific volume size, and can be designed according to the requirement.

Wherein, annotate appearance hole 13, cavity 11 and exhaust hole 12 and be the through-hole, cavity 11 is convenient for make the time the cavity is convenient for follow-up in 11 interior sample's of cavity both sides go on fast rising and falling the temperature for the through-hole cooperates with first seal membrane 2 and second seal membrane 3. The micro-fluidic chip also comprises a cover plate 6, a clamping groove 17 is convexly arranged on the second side of the detection piece body 1, the clamping groove 17 is positioned in the middle of the second side of the detection piece body 1, and the cover plate 6 is buckled and sealed on the clamping groove 17 of the detection piece body 1 through a sealing rubber sheet 7 so as to seal the sample injection hole 13. Wherein, the edge on the slot 17 is provided with a convex clamping structure, so as to facilitate the positioning and installation of the cover plate 6. Through the sealed dual fixed assembly that realizes apron and the draw-in groove 17 lock joint of detecting piece body 1 and sealed film 7, prevent that the sample from spilling over and the revealing of going up and down temperature reaction process.

In practice, the detection reagents (primer probe mixture) are pre-embedded and dried in the chamber 11 before the sample is injected. After the sample to be detected is injected from the injection hole 13 of the detection piece body 1, the sample flows into each chamber 11, and the air in the chamber 11 is exhausted through the exhaust hole 12 so that no bubble is in the chamber 11 to improve the detection result of PCR. The first sealing film 2 and the second sealing film 3 are respectively attached to the first side and the second side of the detection piece body 1 in a bubble-free sealing manner through a heat sealing process, so that the reagent or the sample is prevented from overflowing in the thermal reaction process and the like. Because the first sealing film 2 is arranged on the first side of the vent hole 12 and the hydrophobic breathable film 4 is arranged on the second side of the vent hole 12, the effect that the vent hole 12 can ventilate but is not watertight is achieved. After the pathogen sample to be detected is injected from the sample injection hole 13 of the detection sheet body 1, the cover plate 6 is buckled and sealed on the clamping groove 17 of the detection sheet body 1 to seal the sample injection hole 13 and the exhaust hole 12. Because the cover plate 6 is matched with the detection plate body 1 to seal the sample injection hole 13, the interior of the chamber 11 of the microfluidic chip after sample injection is sealed, the leakage of a sample and a reagent can not be caused in the temperature rise and fall process of PCR reaction, and the environmental pollution is caused, so that the whole chamber 11 can be filled with the sample and the liquid leakage can be avoided while no bubble is generated in the chamber 11 during sample injection.

According to multiple experiments, when equal amount of samples are respectively filled into a circular chamber, an oval chamber, a rectangular chamber and a crescent chamber with the same volume in the same experiment environment, under the observation of a microscope, the average number of bubbles in the circular chamber is more than 2, the average number of bubbles in the oval chamber is 1.4, the average number of bubbles in the rectangular chamber is 8, and the average number of bubbles in the crescent chamber is 0.

When the horizontal cross section of the cavity 11 is of a crescent structure, when liquid flows into the cavity 11, the liquid inlet side of the cavity 11 is gradually expanded, the gas outlet side of the cavity 11 is gradually reduced, so that the liquid is filled while gas is completely discharged without residual air to form bubbles, and the accuracy of a subsequent optical detection result is improved. Meanwhile, the width of each chamber 11 in the optical detection device, i.e. the direction perpendicular to the arrangement of the plurality of chambers 11, cannot exceed a specified size, and the number of chambers 11 can be maximized on the detection sheet body 1 with the same size due to the crescent-shaped structure of the chambers 11 in the invention, i.e. PCR detection of more respiratory pathogens is realized. Chamber 11 is crescent structure, and it includes a concave arc face and convex arc face, with its convex arc face towards the neighboring that detects body 1, this structure is favorable to the refraction gathering of fluorescence to have improved the accuracy of follow-up optical detection result, and in this embodiment, because two upper and lower sides of chamber 11 are as the heating surface simultaneously, heat transfer area is big, and it is faster and even to transfer heat. The chambers 11 are all arranged at the edge of the microfluidic chip, so that the optical detection module arranged at the periphery of the microfluidic chip conveniently enhances optical signals by utilizing the light condensation effect of the arc-shaped surface of the chamber 11, and further facilitates the subsequent optical detection module to collect fluorescence signals. It should be noted that the structure of the chamber 11 can be replaced by an oval structure, a spindle structure, etc., but the convex arc surfaces are preferably oriented toward the peripheral side of the detection sheet body 1.

The shape of the detection sheet body 1 is designed according to actual requirements, and the detection sheet body 1 can be made of transparent materials, preferably polypropylene, polycarbonate, polystyrene or polymethyl methacrylate. And because the micro-fluidic chip in this application, encapsulate the detection reagent in chamber 11 in advance, can use the pipettor to detect the sample injection micro-fluidic chip, the sample gets into chamber 11 through each runner, mixes with pre-sealing reagent, only needs once to extract and detects the sample, the operating personnel's that significantly reduces moves liquid the number of times.

The chamber 11 is a crescent structure, the middle part of which is wider and the two ends of which are gradually reduced, so that the inlet end of the liquid reagent is gradually expanded and the outlet end of the gas is gradually reduced. The gas in the cavity can be discharged when liquid enters the cavity, if the air is not discharged in time, bubbles are easily formed and remained, the gradually-expanding and gradually-reducing structure is used for avoiding forming a right angle, an acute angle or other parts which obstruct the flow of the fluid in the cavity, and the liquid can conveniently enter the cavity and the air in the cavity is discharged.

As shown in fig. 21-22, the first side of the test piece body 1 extends to the second side of the test piece body 1 to form a main flow channel 14 and a branch sample injection flow channel 15, that is, the main flow channel 14, the branch sample injection flow channel 15 and the exhaust flow channel 16 are only hollowed out at one side of the test piece body 1, so as to facilitate the installation of the hydrophobic breathable film 4 on the subsequent exhaust hole 12 of the test piece body 1. The sample injection holes 13 are communicated with a main flow channel 14, each chamber 11 is communicated with the main flow channel 14 through a branch sample injection flow channel 15, and the chambers 11 are communicated with the exhaust holes 12 through exhaust flow channels 16. In this application, the end of the exhaust channel 16 close to the exhaust hole 12 is a slope structure, and then the liquid flows into the exhaust hole 12 under the guidance of a certain slope, and a vortex is formed to discharge bubbles of the liquid. It should be noted that the inner walls of the total flow channel 14, the branch sample introduction flow channel 15 and the exhaust flow channel 16 are smooth transition channels to facilitate the flow of the sample during sample introduction. As shown in fig. 23, the chamber 11 is a through hole on both sides so as to hold more reagents in a small volume range and to quickly and instantly heat both sides, but the branch sample inlet channel 15 and the exhaust channel 16 which are communicated with the chamber 11 are both not penetrated through the detection sheet body 1, and a gentle slope transition 110 is provided at a connection with the chamber 11. The structure is beneficial to the reagent to rapidly enter the cavity 11, reduces turbulence and residual quantity of air bubbles in the cavity 11, and can save the dosage of the reagent. The chamber 11 is a through hole, and is matched with the first sealing film 2 and the second sealing film 3 to carry out double-side heating, so that the samples in the chamber 11 can be rapidly heated and cooled, the temperature gradient formed in the liquid of the chamber 11 caused by single-side heating is avoided, and the reaction speed and the reaction efficiency are improved.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A micro-fluidic chip for detecting nucleic acid of respiratory pathogens is characterized by comprising a plurality of independent chambers, wherein each chamber is respectively communicated with a sample injection hole and an exhaust hole; the number of the chambers is more than or equal to 3;

the other chambers are each independently coated with upstream and downstream primers and probes for detecting one or more of the following pathogen genes: novel coronavirus N gene, novel coronavirus 1ab gene, coronavirus 229E type, coronavirus OC43 type, coronavirus NL63 type, coronavirus HKU1 type, adenovirus general purpose, respiratory syncytial virus A type, respiratory syncytial virus B type, human metapneumovirus, rhinovirus, influenza A virus, influenza B virus, human parainfluenza virus 1 type, human parainfluenza virus 2 type, human parainfluenza virus 3 type, human parainfluenza virus 4 type, and Mycoplasma pneumoniae.

2. The microfluidic chip for detecting nucleic acid of a respiratory pathogen according to claim 1, wherein the upstream and downstream primers for detecting GAPDH are as shown in SEQ ID NO:55 and SEQ ID NO:56, the probe is shown as SEQ ID NO: shown as 57; the upstream and downstream primers for detecting Bacillus atrophaeus DNA are shown as SEQ ID NO:58 and SEQ ID NO:59, and the probe is shown as SEQ ID NO: shown at 60.

3. The microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, wherein the nucleic acid detection chip comprises a first chip and a second chip,

upstream and downstream primers for detecting influenza A virus are shown as SEQ ID NO 1 and SEQ ID NO 2, and a probe is shown as SEQ ID NO 3;

the upstream and downstream primers for detecting the influenza B virus are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the probe is shown as SEQ ID NO. 6;

upstream and downstream primers for detecting the human parainfluenza virus type 1 are shown as SEQ ID NO. 7 and SEQ ID NO. 8, and a probe is shown as SEQ ID NO. 9;

upstream and downstream primers for detecting the respiratory syncytial virus B type are shown as SEQ ID NO. 37 and SEQ ID NO. 38, and a probe is shown as SEQ ID NO. 39;

the upstream and downstream primers for detecting Mycoplasma pneumoniae are shown as SEQ ID NO. 40 and SEQ ID NO. 41, and the probe is shown as SEQ ID NO. 42;

the upstream and downstream primers for detecting rhinovirus are shown as SEQ ID NO. 46 and SEQ ID NO. 47, and the probe is shown as SEQ ID NO. 48;

the upstream and downstream primers for detecting the N gene of the novel coronavirus are shown as SEQ ID NO. 49 and SEQ ID NO. 50, and the probe is shown as SEQ ID NO. 51;

4. The microfluidic chip for detecting nucleic acid of a respiratory pathogen according to claim 1, wherein at least 1 chamber of the plurality of chambers is empty and is not coated with any detection reagent.

5. The microfluidic chip for detecting nucleic acid of a respiratory pathogen according to claim 1, wherein in the process of coating primers and probes in each chamber, a 1uL primer group and probe mixture is spotted into the corresponding chamber by using a pipettor, air-dried, and then a coating film in the chamber is sealed; wherein, in the mixture of the primer group and the probe, the total concentration of the primer group and the probe is 0.4-1 mu mol/L.

6. The microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, wherein the number of chambers is not less than 20.

7. The microfluidic chip for detecting the nucleic acid of the respiratory pathogen according to claim 1, wherein the microfluidic chip comprises a detection sheet body, the detection sheet body is a sheet-shaped plate body and comprises a first side surface and a second side surface, a plurality of chamber holes, a branch sample injection flow channel communicated with a first end of each chamber hole, an exhaust flow channel communicated with a second end, an exhaust hole communicated with each exhaust flow channel, a main flow channel communicated with each branch sample injection flow channel, and a sample injection hole positioned at the starting end of the main flow channel are concavely formed on the first side surface, and the chamber holes are in a through hole structure; the branch sample injection flow passage, the exhaust flow passage and the exhaust hole are of non-through hole structures;

the opening side of the exhaust hole is covered with a hydrophobic breathable film, and a first sealing sheet is compounded on the first side surface of the detection sheet body to block the branch sample injection flow passage, the exhaust flow passage and the first side of the cavity hole; the second side surface of the detection sheet body is compounded with a second sealing sheet so as to seal the second side of the cavity hole, so that the cavity hole becomes a sealed cavity;

8. The microfluidic chip for detecting the nucleic acid of the respiratory pathogen according to claim 1, wherein the horizontal section of the chamber is crescent-shaped, oval-shaped or spindle-shaped, the width of each chamber is gradually narrowed from the middle to the two ends, and the chamber has a convex arc surface; the convex cambered surface is opposite to the peripheral edge facing the detection sheet body.

9. The microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, wherein the length-to-width ratio of the chamber is between 3:1.4-2, length-to-thickness ratio between 3; the volume of the chamber is 5-10 muL.

10. A method for detecting nucleic acid of respiratory pathogens, which is characterized in that the microfluidic chip of any one of claims 1 to 9 is used for detecting nucleic acid, and the detection steps are as follows: