CN115725387A

CN115725387A - Integrated multiple detection kit for digestive tract disease pathogens

Info

Publication number: CN115725387A
Application number: CN202211264596.7A
Authority: CN
Inventors: 赵怀; 封帆; 华灿忠; 黄飞; 查丽虹; 许静文; 羊安钦
Original assignee: Hangzhou Lifereal Biotechnology Co ltd
Current assignee: Hangzhou Lifereal Biotechnology Co ltd
Priority date: 2022-10-17
Filing date: 2022-10-17
Publication date: 2023-03-03

Abstract

The invention relates to the technical field of biological medicines, and discloses an integrated multiple detection kit for pathogens of digestive tract diseases, which comprises a kit body, wherein a rotary plunger, a sample adding bin, a mixing bin, a plurality of liquid storage bins and a plurality of PCR reaction bins are arranged in the kit body; the box body is provided with an air pressure driving module communicated with an air inlet and an air outlet to the mixing bin; the liquid storage bin is sequentially filled with lysis solution, first washing solution, second washing solution, eluent and sealant; a PCR reaction solution is arranged in the PCR reaction bin; the PCR reaction solution comprises a probe primer group; the kit disclosed in the application is suitable for simultaneously realizing detection of 28 pathogen genes in one step, and has the advantages of high sensitivity, strong specificity, short period, high accuracy, rapidness, easiness in operation and the like.

Description

Integrated multiple detection kit for digestive tract disease pathogens

Technical Field

The invention relates to the technical field of biological medicines, in particular to an integrated multiple detection kit for digestive tract disease pathogens.

Background

Digestive tract pathogen infection is a common disease of human beings, greatly influences physical and psychological health of people, has high morbidity and wide range of targeted people, can cause digestive tract infection in all age groups, and has great influence on physical health of patients. Is a human health problem which is very concerned at present domestically and internationally.

The detection and identification technology for the pathogens of the digestive tract diseases, which is commonly used in clinic at present, comprises the following steps: culture method, specific antibody detection, real-time fluorescence PCR method, etc. The novel technology of the integrated gene analysis combining the point-of-care testing (POCT) with the nucleic acid extraction, the real-time fluorescent gene testing and the microfluidic card box only needs manual sample adding, and then the processes of the nucleic acid extraction, the testing and the analysis are completed by a machine, so that the totally-closed automatic output from a sample to a gene testing result is realized, and the novel technology has the characteristics of simple operation, high specificity, low cost, high speed, accurate result and the like. Therefore, real-time fluorescent PCR has become a hot spot for the development of clinical diagnostic systems in recent years.

In the prior art, when a real-time fluorescence PCR method is adopted to detect digestive tract disease pathogens, 1-6 pathogens can be detected at the same time, the detection efficiency is low, and the number of detection items is very limited.

Therefore, how to rapidly and timely detect the pathogens of the digestive tract diseases by using real-time fluorescent PCR becomes a problem to be solved by a plurality of manufacturers.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a multiple detection kit for pathogens of digestive tract diseases.

In a first aspect, the application provides a multiple detection kit for pathogens of digestive tract diseases, which adopts the following technical scheme:

an integrated genital tract disease pathogen multi-connected detection kit comprises a kit body, wherein a rotary plunger, a sample adding bin, a mixing bin, a plurality of liquid storage bins and a plurality of PCR reaction bins are arranged in the kit body, a plurality of different liquid flow channels are arranged on the rotary plunger, and each liquid flow channel is respectively communicated with the sample adding bin, the mixing bin, the liquid storage bins and the PCR reaction bins when the rotary plunger rotates to different angles; the box body is provided with an air pressure driving module communicated with an air inlet and an air outlet to the mixing bin; the liquid storage bin is sequentially filled with lysis solution, first washing solution, second washing solution, eluent and sealant; a PCR reaction solution is arranged in the PCR reaction bin; the PCR reaction solution comprises a probe primer group, and the sequence of the probe primer group is as follows:

the upstream primer of the internal standard comprises a nucleotide sequence shown as SEQ ID NO. 1; the downstream primer of the internal standard comprises a nucleotide sequence shown as SEQ ID NO. 2; the probe of the internal standard comprises a nucleotide sequence shown as SEQ ID NO. 3;

the upstream primer of the Shigella comprises a nucleotide sequence shown as SEQ ID NO. 4; the downstream primer of the Shigella comprises a nucleotide sequence shown as SEQ ID NO. 5; the probe 1 of the Shigella comprises a nucleotide sequence shown as SEQ ID NO. 6; the probe 2 of the Shigella comprises a nucleotide sequence shown as SEQ ID NO. 7;

the upstream primer of the poliovirus comprises a nucleotide sequence shown as SEQ ID NO. 8; the downstream primer of the poliovirus comprises a nucleotide sequence shown as SEQ ID NO. 9; the poliovirus probe comprises a nucleotide sequence shown as SEQ ID NO. 10;

the upstream primer of the salmonella comprises a nucleotide sequence shown as SEQ ID NO. 11; the downstream primer of the salmonella comprises a nucleotide sequence shown as SEQ ID NO. 12; the salmonella probe comprises a nucleotide sequence shown as SEQ ID NO. 13;

the upstream primer of the typhoid bacillus comprises a nucleotide sequence shown as SEQ ID NO. 14; the downstream primer of the typhoid bacillus comprises a nucleotide sequence shown as SEQ ID NO. 15; the typhoid bacillus probe comprises a nucleotide sequence shown as SEQ ID NO. 16;

the upstream primer of the paratyphoid bacillus comprises a nucleotide sequence shown as SEQ ID NO. 17; the downstream primer of the paratyphoid bacillus comprises a nucleotide sequence shown as SEQ ID NO. 18; the probe of the paratyphoid bacillus comprises a nucleotide sequence shown as SEQ ID NO. 19;

the upstream primer of the vibrio cholerae comprises a nucleotide sequence shown as SEQ ID NO. 20; the downstream primer of the vibrio cholerae comprises a nucleotide sequence shown as SEQ ID NO. 21; the probe of the vibrio cholerae comprises a nucleotide sequence shown as SEQ ID NO. 22;

the upstream primer of the Vibrio ElTor comprises a nucleotide sequence shown in SEQ ID NO. 23; the downstream primer of the Vibrio ElTor comprises the nucleotide sequence shown in SEQ ID NO. 24; the probe of the Vibrio ElTor comprises a nucleotide sequence shown in SEQ ID NO. 25;

the upstream primer of the dysentery amoeba comprises a nucleotide sequence shown as SEQ ID NO. 26; the downstream primer of the dysentery amoeba comprises a nucleotide sequence shown as SEQ ID NO. 27; the probe of the dysentery amoeba comprises a nucleotide sequence shown as SEQ ID NO. 28;

the upstream primer of the coxsackie virus comprises a nucleotide sequence shown as SEQ ID NO. 29; the downstream primer of the coxsackie virus comprises a nucleotide sequence shown as SEQ ID NO. 30; the probe of the coxsackie virus comprises a nucleotide sequence shown as SEQ ID NO. 31;

the upstream primer of the enterovirus 71 comprises a nucleotide sequence shown as SEQ ID NO. 32; the downstream primer of the enterovirus 71 comprises a nucleotide sequence shown as SEQ ID NO. 33; the enterovirus 71 probe comprises a nucleotide sequence shown as SEQ ID NO. 34;

the upstream primer of the echovirus comprises a nucleotide sequence shown as SEQ ID NO. 35; the downstream primer of the echovirus comprises a nucleotide sequence shown as SEQ ID NO. 36; the echovirus probe comprises a nucleotide sequence shown as SEQ ID NO. 37;

the upstream primer of the staphylococcus aureus comprises a nucleotide sequence shown as SEQ ID NO. 38; the downstream primer of the staphylococcus aureus comprises a nucleotide sequence shown as SEQ ID NO. 39; the probe of the staphylococcus aureus comprises a nucleotide sequence shown as SEQ ID NO. 40;

the upstream primer of the vibrio parahaemolyticus comprises a nucleotide sequence shown as SEQ ID NO. 41; the downstream primer of the vibrio parahaemolyticus comprises a nucleotide sequence shown as SEQ ID NO. 42; the vibrio parahaemolyticus probe comprises a nucleotide sequence shown as SEQ ID NO. 43;

the upstream primer of the proteus comprises a nucleotide sequence shown as SEQ ID NO. 44; the downstream primer of the proteus comprises a nucleotide sequence shown as SEQ ID NO. 45; the probe of the proteus comprises a nucleotide sequence shown as SEQ ID NO. 46;

the upstream primer of the lumbruous roundworm comprises a nucleotide sequence shown as SEQ ID NO. 47; the downstream primer of the lumbricus ascaris comprises a nucleotide sequence shown as SEQ ID NO. 48; the probe of the lumbruous roundworm comprises a nucleotide sequence shown as SEQ ID NO. 49;

the upstream primer of the cestoda bubbly comprises a nucleotide sequence shown as SEQ ID NO. 50; the downstream primer of the cestoda bubbly comprises a nucleotide sequence shown as SEQ ID NO. 51; the probe of the cestodes bubbly comprises a nucleotide sequence shown as SEQ ID NO. 52;

the Echinococcus upstream primer comprises a nucleotide sequence shown as SEQ ID NO. 53; the Echinococcus hainanensis downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 54; the probe of the echinococcus comprises a nucleotide sequence shown as SEQ ID NO. 55;

the upstream primer of the taenia multiceps comprises a nucleotide sequence shown as SEQ ID NO. 56; the downstream primer of the taenia multiceps comprises a nucleotide sequence shown as SEQ ID NO. 57; the tapeworm polytape probe comprises a nucleotide sequence shown as SEQ ID NO. 58;

the upstream primer of the enterobacter helminthiasis comprises a nucleotide sequence shown as SEQ ID NO. 59; the downstream primer of the enterobacter helminthiasis comprises a nucleotide sequence shown as SEQ ID NO. 60; the probe of the helminthic nematode comprises a nucleotide sequence shown as SEQ ID NO. 61;

the upstream primer of the fasciola brucei comprises a nucleotide sequence shown as SEQ ID NO. 62; the downstream primer of fasciolopsis brunetti comprises a nucleotide sequence shown as SEQ ID NO. 63; the probe of fasciolopsis brunetti comprises a nucleotide sequence shown as SEQ ID NO. 64;

the upstream primer of the lactobacillus sake comprises a nucleotide sequence shown as SEQ ID NO. 65; the downstream primer of the lactobacillus sakei comprises a nucleotide sequence shown as SEQ ID NO. 66; the probe of the lactobacillus sakei comprises a nucleotide sequence shown as SEQ ID NO. 67;

the upstream primer of lactobacillus acidophilus comprises a nucleotide sequence shown as SEQ ID NO. 68; the downstream primer of lactobacillus acidophilus comprises a nucleotide sequence shown as SEQ ID NO: 69; the probe of the lactobacillus acidophilus comprises a nucleotide sequence shown as SEQ ID NO. 70;

the upstream primer of enterococcus faecium comprises a nucleotide sequence shown as SEQ ID NO. 71; the downstream primer of the enterococcus faecium comprises a nucleotide sequence shown as SEQ ID NO. 72; the probe of enterococcus faecium comprises a nucleotide sequence shown as SEQ ID NO. 73;

the upstream primer of the bacteroides fragilis comprises a nucleotide sequence shown as SEQ ID NO. 74; the downstream primer of the bacteroides fragilis comprises a nucleotide sequence shown as SEQ ID NO. 75; the probe of the bacteroides fragilis comprises a nucleotide sequence shown as SEQ ID NO. 76;

the upstream primer of the clostridium botulinum comprises a nucleotide sequence shown as SEQ ID NO. 77; the downstream primer of clostridium botulinum comprises a nucleotide sequence shown as SEQ ID No. 78; the probe of clostridium botulinum comprises a nucleotide sequence shown as SEQ ID NO: 79;

the upstream primer of the streptococcus digestus comprises a nucleotide sequence shown as SEQ ID NO. 80; the downstream primer of the streptococcus digestus comprises a nucleotide sequence shown as SEQ ID NO. 81; the probe for digesting streptococcus comprises a nucleotide sequence shown as SEQ ID NO: 82;

the upstream primer of the clostridium perfringens comprises a nucleotide sequence shown as SEQ ID NO. 83; the downstream primer of the clostridium perfringens comprises a nucleotide sequence shown as SEQ ID NO. 84; the probe of the clostridium perfringens comprises a nucleotide sequence shown as SEQ ID NO. 85;

the upstream primer of the Escherichia coli comprises a nucleotide sequence shown as SEQ ID NO. 86; the downstream primer of the Escherichia coli comprises a nucleotide sequence shown as SEQ ID NO. 87; the probe of Escherichia coli comprises a nucleotide sequence shown as SEQ ID NO: 88.

Wherein, a PCR reaction solution is arranged in the PCR reaction bin; the PCR reaction solution comprises a mixed solution of a probe primer group and a HotStart Onestep RT-PCR Master Mix, and the volume ratio of the probe primer group to the HotStart Onestep RT-PCR Master Mix is (1-3): (3-5).

The HotStart Onestep RT-PCR Master Mix reagent mainly contains HotStart rTth enzyme which is in Mn ²⁺ Under the activated condition, the compound has high-efficiency amplification activity.

The HotStart Onestrep RT-PCR Master Mix is from Hangzhou shinkangyi Biotechnology Inc. and has the product types as follows: B0027.

preferably, the lysis solution comprises 1-1000mM acetic acid-sodium acetate, 0.1-10% by mass of polyethanol, 0.1-10M guanidinium isothiocyanate, 0.1-10% by volume of Triton X-100, and 1-5% by mass of sodium dodecyl sarcosinate.

Preferably, the pH of the lysate is adjusted to 5.0-7.0 with acetic acid.

Preferably, the first washing reagent comprises Tris-HCl 1-500mM (pH 8.0), potassium chloride 10-500mM, guanidinium isothiocyanate 0.1-10M, and 0.1-70% by mass of polyethanol.

Preferably, the pH of the first wash solution is adjusted to 5.0-8.0 with acetic acid.

Preferably, the second washing solution comprises Tris-HCl 1-500mM (pH 8.0), potassium chloride 10-500mM, polyethylene glycol with the mass concentration of 0.1-10% and non-protein blocking agent with the volume concentration of 0.1-5%.

Preferably, the pH of the second washing solution is adjusted to 5.0 to 8.0 with acetic acid.

Preferably, the eluent is 1-50mM Tris-HCl.

Preferably, the blocking agent has good chemical stability, hydrophobicity, density of 0.1-1.5g/ml and viscosity of 1000-200000CSt.

Preferably, the blocking agent may be a silicone oil, such as silicone oil, a silicone base, or other polymeric material.

Preferably, a meltable material layer is arranged in the PCR reaction bin; the meltable material layer is embedded with manganese ion solution.

Preferably, the meltable material is at least one of paraffin, dodecane, tetradecane, hexadecane.

Preferably, the manganese ion solution comprises 0.1-100mM of manganese acetate, 20-60% of trehalose by mass concentration and the balance of ultrapure water; the volume of the manganese ion solution is 5-10ul.

The melting point range of the meltable material is 20-95 ℃, and the meltable material can be stored for a long time at the temperature of 2-8 ℃.

The meltable material can be used for embedding a manganese ion solution in a reaction solution; the manganese ion solution in the reaction solution can be embedded at a low temperature, and the manganese ion solution in the reaction solution can be mixed with the reaction solution after being melted at a high temperature; when in use, the PCR product is melted to form oily substances, so that the PCR product of the aerosol can be prevented from polluting a laboratory.

The invention adopts POCT technology, can detect and analyze 28 digestive tract related pathogen diseases according to the amplification result of the multiple slots, and contains an internal quality control function (internal standard reagent) to control the false positive of the result and the correctness of the sample collection and purification process.

By adopting the technical scheme, the detection of 28 digestive tract disease pathogen genes can be simultaneously realized in one step, and the method has the advantages of high sensitivity, strong specificity, short period, high accuracy, rapidness, easiness in operation and the like; the kit can be widely applied to a plurality of fields of clinical diagnosis of digestive tract diseases, marriage and childbirth instruction, prenatal screening, prenatal and postnatal care, prognosis treatment, scientific research and the like.

In summary, the present application includes at least one of the following beneficial technical effects:

1. according to the kit, the processes of nucleic acid extraction, amplification, detection and the like are concentrated in the totally-enclosed cartridge, so that aerosol pollution is avoided, the safety of detection personnel is protected, and a PCR laboratory is not needed; the problem of equipment pollution caused by nucleic acid extraction is avoided;

2. the kit disclosed in the application is suitable for in-vitro qualitative detection of 28 kinds of digestive tract disease pathogen nucleic acids, can be used for detecting 28 kinds of different pathogens in the same kit at one time, and has the advantages of high sensitivity, strong specificity, short period, high accuracy, rapidness, easiness in operation and the like.

Drawings

FIG. 1 is a schematic structural diagram of an integrated multiple detection kit for pathogens of genital tract diseases;

FIG. 2 is a schematic structural view of a cartridge body;

FIG. 3 is a schematic view of the structure of the rotary plunger;

in the figure: 1. the device comprises a box body, 2, a rotary plunger, 3, a sample adding bin, 4, a mixing bin, 5, a liquid storage bin, 6, a PCR reaction bin, 7, a liquid flow channel, 8, an air hole, 9, an air pressure driving module, 10, an air valve, 11 and a meltable material layer.

Detailed Description

The present application will be described in further detail with reference to examples.

Structure of reagent kit

An integrated genital tract disease pathogen multi-connection detection kit comprises a kit body 1, wherein a rotary plunger 2, a sample adding bin 3, a mixing bin 4, a plurality of liquid storage bins 5 and a plurality of PCR reaction bins 6 are arranged in the kit body 1, a plurality of different liquid flow channels 7 are arranged on the rotary plunger 2, and each liquid flow channel 7 is respectively communicated with the sample adding bin 3, the mixing bin 4, the liquid storage bins 5 and the PCR reaction bins 6 when the rotary plunger 2 rotates to different angles; the box body 1 is provided with an air pressure driving module 9 with an air inlet and an air outlet communicated with the mixing bin 4; respectively adding 8 groups of PCR reaction liquid into the PCR reaction bin 6; lysis solution, first washing solution, second washing solution, eluent and sealant are sequentially filled in the liquid storage bin 5.

Be provided with the application of sample hole on application of sample storehouse 3, mix all to be equipped with the gas pocket 8 of gas business turn over, the liquid hole of liquid business turn over on storehouse 4, the stock solution storehouse 5, be equipped with valve 10 on the box body 1, still be equipped with a plurality of gas flow channels on the rotary plunger 2, every gas flow channel communicates valve 10 and mix storehouse 4's gas pocket 8 under rotary plunger 2 rotates to different angles respectively. When any one of the liquid storage bins 5 is communicated with the mixing bin 4 through the liquid flow passage 7, the liquid storage bin 5 is communicated to the air valve 10 through the air flow passage. The function of the air valve is to enable the air in the functional bin (the liquid storage bin 5 and the like) to freely enter and exit, the pressure in the bin is constant, and when the air pressure driving module 9 is driven to move and the air in the mixing bin 4 is changed, the liquid is transferred along the liquid path according to the pressure difference between the two bins.

The structure of the kit used in the present application is described in the patent: a pneumatic integrated PCR detection kit based on a rotary plunger and a detection method thereof are disclosed as follows: CN 114015567A.

The sample extraction and detection process is as follows:

(1) Sample adding: adding a sample to be detected into the sample adding bin 3;

(2) Cracking: adding magnetic bead liquid into a mixing bin 4, enabling a sample adding bin 3 to be communicated with the mixing bin 4 through rotation of a rotary plunger 2, transferring sample mixed liquid to the mixing bin 4 by utilizing an air pressure driving module 9, rotating the rotary plunger 2 to another angle, enabling a liquid storage bin 5 for storing pyrolysis liquid to be communicated with the mixing bin 4, operating the air pressure driving module 9 to mix the pyrolysis liquid into the mixing bin 4, cracking the sample to release nucleic acid, then using an external magnet to generate magnetic force to adsorb the magnetic beads in the mixing bin 4, standing, and sending supernatant out of the mixing bin 4 after the sample mixed liquid becomes clear;

(3) Cleaning: the rotary plunger 2 is rotated to a specific angle, so that the mixing bin 4 on the box body 1 is sequentially communicated with the liquid storage bin 5 through the liquid flow channel 7, then the air pressure driving module 9 is used for moving to change the pressure of the mixing bin 4, further the first washing liquid and the second washing liquid enter the mixing bin 4, impurities on nucleic acid are washed away to obtain a nucleic acid template solution, and the air pressure driving module 9 is moved reversely to enable a reagent to return to the mixing bin 4, so that one-time or multiple-time cleaning of sample nucleic acid is realized;

(4) And (3) elution: the rotary plunger 2 is rotated to a specific angle, so that the mixing bin 4 on the box body 1 is communicated with the liquid storage bin 5 containing eluent by using the liquid flow channel 7, then the eluent is sucked into the mixing bin 4 by using the movement of the air pressure driving module 9, and the nucleic acid on the magnetic beads is eluted, so that the subsequent PCR reaction is carried out;

(5) And (3) PCR reaction: the rotary plunger 2 is rotated to a specific angle, so that the mixing bin 4 on the box body 1 is communicated with the PCR reaction bin 6 through a liquid flow channel 7, then the air pressure driving module 9 is used for moving to send the eluted nucleic acid eluent into the PCR reaction bin 6, the meltable material is heated to release magnesium ions, the eluted nucleic acid eluent and the magnesium ion solution enter each reaction cavity filled with reaction liquid together to carry out PCR amplification detection, the nucleic acid is subjected to PCR reaction in the reaction liquid in the PCR reaction bin 6, and the upper part of a channel of the PCR reaction bin 6 is sealed by the sealing agent to avoid mutual influence.

The magnetic microsphere suspension in the card box can move back and forth in the lysis solution, the first washing solution, the second washing solution and the reaction solution.

Example 1: preparation of the kit

1. Designing a primer probe:

the kit of the invention can be used for detecting 28 digestive tract disease pathogens: shigella (SC), poliovirus (PV), salmonella (SAL), typhoid bacillus (ST), paratyphoid Bacillus (BP), vibrio Cholerae (VC), vibrio ELTOR (ELTOR), amebic dysentery (EH), coxsackie virus (CA 16), enterovirus type 71 (EV 71), echovirus (EV), staphylococcus Aureus (SA), vibrio Parahaemolyticus (VP), proteus (PV), ascaridium Lumbricus (AL), tapeworm (TH), echinococcus (EGM), tapeworm (TM), enterohelminth (ETVM), fasciola Brunetti (FB), lactobacillus sake (LACS), lactobacillus acidophilus (LACA), enterococcus faecium (ETF), bacteroides Fragilis (BF), clostridium Botulinum (CB), streptococcus digestus (PTTT), clostridium Perfringens (CP), escherichia coli (E.C).

The invention designs primers and probes aiming at 28 digestive tract disease pathogens, selects 28 digestive tract disease pathogens as target genes of the kit, and designs the primers and the corresponding probes aiming at each pathogen respectively, wherein the sequences are as shown in the following table 1:

TABLE 1 Probe primer sequence Listing

2. Purifying the reagent components:

2.1, preparing liquid

Lysis solution: 500mM acetic acid-sodium acetate, 5% by mass of polyethanol, 5M guanidinium isothiocyanate, 4% by volume of Triton X-100, and 3% by mass of sodium lauryl sarcosine.

The pH of the lysate was adjusted to 6.0 with acetic acid.

A first washing solution: tris-HCl 300mM (pH8.0), potassium chloride 250mM, guanidinium isothiocyanate 5M, and 30% by mass of ethanol.

The pH of the first washing reagent was adjusted to 6.0 with acetic acid.

A second washing solution: tris-HCl 400mM (pH8.0), potassium chloride 300mM, polyethylene glycol with the mass concentration of 5% and non-protein blocking agent with the volume concentration of 3%.

The pH of the second wash was adjusted to 7.0 with acetic acid.

Wherein the non-protein blocking agent is purchased from Biotechnology engineering (Shanghai) Inc. with the model number of NO. C530040.

Eluent: 10mM Tris-HCl (pH 8.0).

A sealing agent: a silicone oil.

Meltable material: paraffin wax.

Manganese ion solution 7. Mu.l: comprises 45mM of manganese acetate, trehalose with the mass concentration of 40 percent, and the balance of the trehalose is supplemented with sterilized ultrapure water.

PCR reaction solution: a mixture of 25. Mu.l of HotStart Onestep RT-PCR Master Mix solution and 10. Mu.l of primer probe solution (containing water) was prepared, wherein the final concentration of each primer in the primer probe solution was 0.5. Mu.M and the concentration of each probe was 0.25. Mu.M.

2.2, assembling the kit: packaging the above components into kit, and sealing each reagent with appropriate amount of silicone oil.

3. Description of the experiments

3.1 sample Collection and processing

(1) Sample preparation: a fecal swab;

(2) Sample treatment:

adding 1ml of physiological saline into a sampling tube, fully oscillating, extruding and washing a swab in the sampling tube, and taking out the swab to obtain eluent; if there is more suspension with large particles in the liquid, centrifuging at low speed (3000rpm, 2min), and collecting supernatant (200 μ l);

mu.l of the eluate (or supernatant) was taken to the

sample application compartment

3 and 10. Mu.l of magnetic beads was added.

3.2, putting the kit into a machine for automatic operation after detection, wherein the reaction program is shown in table 2:

TABLE 2 reaction procedure

3.3, test results

(1) After the operation of the detection program is finished, the detection result is automatically reported by an applicable instrument.

(2) The results show the detection results of the target and the internal quality control.

(3) The result shows that the target gene result should present a typical S-type amplification curve (including an S curve before the plateau), and Ct is less than or equal to 38, and the target gene result is judged to be positive to the pathogen.

(4) The internal quality control (internal standard) fluorescence channel detection result should present a typical S-shaped amplification curve (including an S-curve before the plateau stage). When the target is negative and the internal quality control Ct is less than or equal to 38, the experimental result is effective. Otherwise, it needs to be re-sampled and detected.

3.4, the detection results of the samples are shown in Table 3.

TABLE 3 results of sample testing

The invention can simultaneously detect more than 10 (28) digestive tract pathogen nucleic acids in one detection unit (card box) at one time, and has internal quality control function to control the false positive of the result and the correctness of the sample collection and purification process; has the advantages of simple and convenient operation, high sensitivity, simultaneous detection of various types and the like.

The application designs special primers and probes aiming at 28 digestive tract pathogen nucleic acid specific sequences, ensures the specificity and sensitivity of detection, and is widely applied to multiple fields of digestive tract disease clinical diagnosis, marriage and education guidance, prenatal screening, prenatal and postnatal care, prognostic treatment, scientific research and the like.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims

1. A multiple detection kit for pathogens of digestive tract diseases is characterized in that: the device comprises a box body (1), wherein a rotary plunger (2), a sample adding bin (3), a mixing bin (4), a plurality of liquid storage bins (5) and a plurality of PCR reaction bins (6) are arranged in the box body (1), a plurality of liquid flow channels (7) are arranged on the rotary plunger (2), and each liquid flow channel (7) is respectively communicated with the sample adding bin (3), the mixing bin (4), the liquid storage bins (5) and the PCR reaction bins (6) when the rotary plunger (2) rotates to different angles; the box body (1) is provided with an air pressure driving module (9) with an air inlet and an air outlet communicated with the mixing bin (4); the liquid storage bin (5) is sequentially filled with lysis solution, first washing solution, second washing solution, eluent and sealing agent; a PCR reaction solution is arranged in the PCR reaction bin (6); the PCR reaction solution comprises a probe primer group, and the sequence of the probe primer group is as follows:

the upstream primer of poliovirus comprises a nucleotide sequence shown as SEQ ID NO. 8; the downstream primer of the poliovirus comprises a nucleotide sequence shown as SEQ ID NO. 9; the poliovirus probe comprises a nucleotide sequence shown as SEQ ID NO. 10;

the upstream primer of the staphylococcus aureus comprises a nucleotide sequence shown as SEQ ID NO. 38; the downstream primer of the staphylococcus aureus comprises a nucleotide sequence shown as SEQ ID NO. 39; the staphylococcus aureus probe comprises a nucleotide sequence shown as SEQ ID NO. 40;

the upstream primer of the lumbruous roundworm comprises a nucleotide sequence shown as SEQ ID NO. 47; the downstream primer of the lumbruous roundworm comprises a nucleotide sequence shown as SEQ ID NO. 48; the probe of the lumbruous roundworm comprises a nucleotide sequence shown as SEQ ID NO. 49;

the upstream primer of the lactobacillus sake comprises a nucleotide sequence shown as SEQ ID NO. 65; the downstream primer of the lactobacillus sakei comprises a nucleotide sequence shown as SEQ ID NO. 66; the probe of the lactobacillus sake comprises a nucleotide sequence shown as SEQ ID NO. 67;

the upstream primer of lactobacillus acidophilus comprises a nucleotide sequence shown as SEQ ID NO. 68; the downstream primer of lactobacillus acidophilus comprises a nucleotide sequence shown as SEQ ID NO: 69; the lactobacillus acidophilus probe comprises a nucleotide sequence shown as SEQ ID NO. 70;

the upstream primer of the enterococcus faecium comprises a nucleotide sequence shown as SEQ ID NO. 71; the downstream primer of the enterococcus faecium comprises a nucleotide sequence shown as SEQ ID NO. 72; the probe of enterococcus faecium comprises a nucleotide sequence shown as SEQ ID NO. 73;

the upstream primer of the clostridium botulinum comprises a nucleotide sequence shown as SEQ ID NO. 77; the downstream primer of the clostridium botulinum comprises a nucleotide sequence shown as SEQ ID NO. 78; the probe of clostridium botulinum comprises a nucleotide sequence shown as SEQ ID NO: 79;

2. The multiple detection kit for pathogens of digestive tract diseases according to claim 1, wherein the multiple detection kit comprises: the PCR reaction solution also comprises a HotStart Onestep RT-PCR Master Mix, and the volume ratio of the probe primer group to the HotStart Onestep RT-PCR Master Mix (1-3): (3-5).

3. The multiple detection kit for pathogens of digestive tract diseases according to claim 1, wherein the multiple detection kit comprises: the lysis solution comprises 1-1000mM acetic acid-sodium acetate, 0.1-10% of polyethanol with mass concentration, 0.1-10M guanidinium isothiocyanate, 0.1-10% Triton X-100 with volume concentration and 1-5% sodium dodecyl sarcosine with mass concentration.

4. The multiple detection kit for pathogens of digestive tract diseases according to claim 1, wherein the multiple detection kit comprises: the first washing solution comprises Tris-HCl 1-500mM, potassium chloride 10-500mM, guanidinium isothiocyanate 0.1-10M and 0.1-70% of polyethanol by mass concentration.

5. The multiple detection kit for pathogens of digestive tract diseases according to claim 1, wherein the multiple detection kit comprises: the second washing solution comprises Tris-HCl 1-500mM, potassium chloride 10-500mM, polyethylene glycol with the mass concentration of 0.1-10% and a non-protein blocking agent with the volume concentration of 0.1-5%.

6. The multiple detection kit for pathogens of digestive tract diseases according to claim 1, wherein the multiple detection kit comprises: the density of the sealant is 0.1-1.5g/ml, and the viscosity is 1000-200000CSt.

7. The multiple detection kit for pathogens of digestive tract diseases according to claim 1, wherein the multiple detection kit comprises: the eluent is 1-50mM Tris-HCl.

8. The multiple detection kit for pathogens of digestive tract diseases according to claim 1, wherein the multiple detection kit comprises: a meltable material layer is arranged in the PCR reaction bin (6); the meltable material layer is embedded with manganese ion solution.

9. The multiple detection kit for pathogens of digestive tract diseases according to claim 8, wherein the multiple detection kit comprises: the meltable material is at least one of paraffin, dodecane, tetradecane and hexadecane.

10. The multiple detection kit for pathogens of digestive tract diseases according to claim 8, wherein the multiple detection kit comprises: the manganese ion solution comprises 0.1-100mM of manganese acetate and 20-60% of trehalose by mass concentration.