CN106916906B - Primer composition for detecting infectious diarrhea pathogens and kit thereof - Google Patents
Primer composition for detecting infectious diarrhea pathogens and kit thereof Download PDFInfo
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Abstract
The invention relates to a primer composition for detecting infectious diarrhea pathogens, which comprises at least one group of rotavirus primer group, enterovirus primer group, norovirus primer group, salmonella primer group, shigella primer group and campylobacter jejuni primer group; also relates to a kit containing the primer composition, which also comprises a micro-fluidic chip coated with the primer, isothermal amplification reaction liquid, isothermal amplification enzyme solution and a negative control substance; also relates to a detection method adopting the primer composition, which comprises coating of the primer composition, nucleic acid extraction of a sample to be detected, LAMP reaction and result interpretation. The kit provided by the invention can be used for quickly and accurately detecting infectious diarrhea pathogens within 1 hour, and has important significance for quickly and auxiliarily guiding treatment and medication; the multi-index detection can also be used for regional epidemiological investigation and epidemic situation monitoring so as to research the epidemic situation of infectious diarrhea in China.
Description
Technical Field
The invention belongs to the technical field of nucleic acid amplification, and particularly relates to a primer composition for detecting infectious diarrhea pathogens, a kit for detecting infectious diarrhea pathogens based on a microfluidic chip system, and a method thereof.
Background
Infectious diarrhea is the most common disease worldwide, and it is estimated that 320 ten thousand children with the age less than or equal to 5 die of diarrhea every year worldwide, and accounts for 24.8% of the death cause of the children of the same age; infectious diarrhea remains an important disease of the current group, as seen in developing countries, which suffer from diseases 1-12 times per child per year, developed countries, and 1-5 times per child per year. Infectious diarrhea refers to diarrhea caused by intestinal inflammation due to various acute and chronic bacterial, viral, fungal and parasitic infections. Infectious diarrhea, other than cholera, bacillary and amebic dysentery, typhoid and paratyphoid, is also called infectious diarrhea, and is a class c infectious disease prescribed in the infectious disease prevention and treatment law of the people's republic of China.
The pathogens of infectious diarrhea include bacteria, viruses, parasites, fungi, etc. At present, bacteria and viruses are mainly used in China, the viruses comprise rotavirus, norovirus, adenovirus, coxsackie virus and the like, and the bacteria mainly comprise salmonella, shigella, campylobacter jejuni and the like; the reports show that the viral infection accounts for about 90 percent of infectious diarrhea and the bacteria accounts for about 10 percent; more than 95% of the viruses are caused by rotavirus and (or) adenovirus and norovirus, and the bacteria mainly comprise salmonella, campylobacter jejuni and shigella, accounting for more than 90%. Infectious diarrhea is one of the most common diseases in China clinically, and if severe patients cannot take the medicine timely and correctly, life is easily threatened. However, the clinical routine analysis of biochemical indicators does not allow accurate identification of the pathogens that a patient is infected with, and most clinical treatments are still in the empirical phase with great blindness in the use of higher antibiotics. The current methods for identifying pathogens that are used in clinical practice mainly rely on bacterial culture assays, which have major drawbacks: the detection time is long (generally about 2-3 days), the accuracy is low (the false negative rate and the false positive rate are both high), and the pathogen which is difficult to culture is difficult to detect.
LAMP (loop-mediated isothermal amplification) is a constant-temperature nucleic acid amplification method developed in 2000, and is characterized in that 4 specific primers are designed for 6 regions of a target gene, and a strand displacement DNA polymerase is used for heat preservation for 30-60 minutes under an isothermal condition (60-65 ℃) so as to complete a nucleic acid amplification reaction. The LAMP has the advantages of simplicity, rapidness, sensitivity and specificity, and is particularly suitable for application and popularization of the LAMP kit in the basement layer. In the LAMP technology, primers are key factors for determining the sensitivity and specificity of detection results.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a primer composition for detecting infectious diarrhea pathogens and a kit thereof, so as to quickly and accurately identify the types of the infectious diarrhea pathogens.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides a primer composition for detecting infectious diarrhea pathogens, comprising at least one of a rotavirus primer set, an enterovirus primer set, a norovirus primer set, a salmonella primer set, a shigella primer set and a campylobacter jejuni primer set;
wherein the rotavirus primer group comprises SEQ ID NO: 1, rotavirus primer F3 shown in SEQ ID NO: 2, rotavirus primer B3 shown in SEQ ID NO: 3 and the rotavirus primer FIP shown in SEQ ID NO: 4, rotavirus primer BIP;
wherein the intestinal adenovirus primer group comprises SEQ ID NO: 5, and the primers F3 and SEQ ID NO: 6, and the primers B3 of the enteroadenovirus shown in SEQ ID NO: 7 and the primers FIP and SEQ ID NO: 8, an enteroadenovirus primer BIP;
wherein the norovirus primer set comprises SEQ ID NO: 9, norovirus primer F3, SEQ ID NO: 10, norovirus primer B3, SEQ ID NO: 11 and norovirus primer FIP as shown in SEQ ID NO: 12 of norovirus
Wherein the salmonella primer set comprises SEQ ID NO: 13, salmonella primer F3, SEQ id no: 14, salmonella primer B3, SEQ ID NO: 15 and the salmonella primer FIP shown in SEQ ID NO: 16, BIP as a salmonella primer;
wherein the Shigella primer set comprises SEQ ID NO: 17, shigella primer F3, SEQ id no: 18, shigella primer B3, SEQ ID NO: 19 and the shigella primer FIP shown in SEQ ID NO: 20, shigella primer BIP;
wherein the campylobacter jejuni primer group comprises SEQ ID NO: 21, and a campylobacter jejuni primer F3 shown in SEQ ID NO: 22, and a campylobacter jejuni primer B3 shown in SEQ ID NO: 23 and primers FIP of campylobacter jejuni shown in SEQ ID NO: 24, and campylobacter jejuni primer BIP.
In order to further optimize the above technical solution, the technical measures taken by the present invention further include:
preferably, the primer composition further comprises an internal control primer group, wherein the internal control primer group is used for amplifying human hemoglobin- β chain (HBB);
wherein the internal control primer group comprises SEQ ID NO: 25, internal control primer F3 shown in SEQ ID NO: 26, internal control primer B3 shown in SEQ ID NO: 27 and the internal control primer FIP shown in SEQ ID NO: 28 as shown in figure 28.
The primer sequences of the infectious diarrhea pathogen primer set and the sequences of the internal control primer set are shown in table 1.
TABLE 1 sequence Listing of amplification primers
On the other hand, the invention provides a kit containing the primer combination, which further comprises a microfluidic chip, wherein the microfluidic chip is provided with 4 reaction detection areas which are not communicated with each other, each reaction detection area comprises a sample injection pool, a distribution pool, a capillary micro valve and an amplification pool which are sequentially communicated, each reaction detection area is provided with 8 amplification pools, and each primer component in the primer combination is respectively coated in the corresponding amplification pool.
In order to further optimize the above technical solution, the technical measures taken by the present invention further include:
preferably, the microfluidic chip is a disc-shaped microfluidic chip.
Preferably, in each amplification pool, the concentration of the primer F3 in each primer group is 1-4 μ M, the concentration of the primer B3 is 1-4 μ M, the concentration of the primer FIP is 8-15 μ M, and the concentration of the primer BIP is 8-15 μ M.
Preferably, the kit further comprises a constant temperature amplification reaction solution, a constant temperature amplification enzyme solution and a negative control substance.
Preferably, the isothermal amplification reaction solution comprises 200-250 mM Tris-HCl, 300-400 mM KCl, 8-10 mM dNTP, 100-120 mM MgSO4, 1-5% (W/V) Tween-20, 40-50% (W/V) glycerol, 1-5% (W/V) PEG8000 and 10-20 mM indicator; the isothermal amplification enzyme solution comprises 10-12U/mu L of DNA polymerase and 20-30U/mu L of reverse transcriptase.
Preferably, the isothermal amplification reaction solution comprises 250mM Tris-HCl, 300mM KCl, 10mM dNTP, 100mM MgSO4, 5% (W/V) Tween-20, 50% (W/V) glycerol, 5% (W/V) PEG8000 and 10mM indicator.
Preferably, the indicator comprises HNB, Calcein, cresol red, phenol red, m-cresol purple, bromocresol purple, neutral red, naphtholphthalein, thymol blue.
Preferably, the indicator is neutral red with the concentration of 10-20 mM, and more preferably 10 mM. Neutral red (Neutralred), known by the scientific name of 3-amino-7-methylamino-2-methylphenazine hydrochloride, is a weakly alkaline pH indicator, has a color change range of pH 6.4-8.0 (changing from red to yellow), is red under acidic conditions, and is yellow under alkaline conditions; before the isothermal amplification reaction, the reaction solution is alkaline, so the color is yellow, and when the amplification reaction occurs, the pH environment of the solution is changed into acid along with the continuous accumulation of amplification products, so the color is changed from yellow to red.
Preferably, the DNA polymerase is Bst DNA polymerase and the reverse transcriptase is M-MLV reverse transcriptase; more preferably Bst DNA polymerase at a concentration of 10U/. mu.L and M-MLV reverse transcriptase at a concentration of 20U/. mu.L.
Preferably, the negative control is rnase-free water.
Finally, the present invention also provides a method for the non-diagnostic purpose of detecting infectious diarrhea pathogens using the above primer composition, comprising the steps of:
step 1) coating of primer composition: coating each primer group in the primer composition in a corresponding amplification pool respectively, and fixing the primer group in the amplification pool through vacuum drying;
step 2) extracting nucleic acid of a sample to be detected: extracting nucleic acid of a sample to be detected by adopting a magnetic bead method;
step 3), LAMP reaction: mixing the constant temperature amplification reaction solution, DNA polymerase and reverse transcriptase solution with the nucleic acid of the sample to be detected, transferring to a sample injection pool of a micro-fluidic chip coated with a primer group, allowing the liquid to flow into the amplification pool under the action of centrifugal force, and then performing loop-mediated constant temperature amplification reaction;
step 4), interpretation of results: and (4) carrying out direct visual interpretation or adopting equipment to carry out color interpretation according to the color change in the amplification pool after the amplification is finished.
Preferably, the coating of the primer composition in step 1) comprises the following specific steps: mixing each primer group of the primer composition with sucrose to prepare a corresponding mixed solution, wherein the final concentrations of the primer group and the sucrose in the mixed solution are 0.15 mu M and 1.0 percent (mass percentage), respectively; dropping 1 μ L of the mixed solution into corresponding amplification pools of the microfluidic chip, drying in a drying oven at 37 ℃, tabletting and sealing a membrane, and coating the primer group in the corresponding amplification pool after punching and vacuumizing; more specifically, the following primer groups are respectively coated on 8 amplification pools of each reaction detection area in the microfluidic chip: a rotavirus LMAP primer group, an enteroadenovirus LAMP primer group, a norovirus LAMP primer group, a salmonella LAMP primer group, a shigella LAMP primer group, a campylobacter jejuni LAMP primer group, a negative control product and an internal control primer group.
Preferably, the specific steps of the LAMP reaction in step 3) are as follows: taking 25 mu L of constant-temperature amplification reaction liquid, 10 mu L of LDNA polymerase and 10 mu L of reverse transcriptase solution, mixing with 40 mu L of nucleic acid of a sample to be detected, transferring to a sample injection pool of a coating primer group microfluidic chip, allowing the liquid to flow into the amplification pool under the action of centrifugal force in the centrifuge, reacting at a constant temperature of 50 ℃ for 10 minutes, and reacting at a constant temperature of 60-63 ℃ for 50 minutes; wherein the isothermal amplification reaction solution, the DNA polymerase and the reverse transcriptase solution all adopt the reagents in the kit.
Preferably, the direct visual interpretation of step 4) is as follows: if the color changes, the sample to be detected contains infectious diarrhea pathogen genome nucleic acid; if the color is not changed, the sample to be detected does not contain infectious diarrhea pathogen genome nucleic acid.
Preferably, the sample to be tested comprises feces; correspondingly, the nucleic acid of the test sample comprises DNA or RNA extracted from the feces.
The application of the primer group or the primer group composition or the kit in the detection or the auxiliary detection of infectious diarrhea pathogens for non-diagnostic purposes also belongs to the protection scope of the invention.
In the present invention, the infectious diarrhea pathogen may be at least one of: rotavirus, enteroadenovirus, norovirus, salmonella, shigella and campylobacter jejuni.
Compared with the prior art, the invention has the following beneficial effects:
the kit provided by the invention provides a high-throughput detection method, can quickly and accurately detect common infectious diarrhea pathogen infection, can obtain detection results of 6 pathogen indexes within 1 hour for clinic, is faster than a real-time fluorescent quantitative PCR method generally adopted at present, and has important significance for quick auxiliary guidance treatment and medication; meanwhile, the detection of multiple indexes can also be used for regional epidemiological investigation and epidemic situation monitoring so as to research the epidemic situation of infectious diarrhea in China.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic chip employed in the present invention;
FIGS. 2 and 3 are schematic diagrams illustrating the coating of a primer set in a microfluidic chip according to an embodiment of the present invention;
FIGS. 4 and 5 are graphs showing the results of detection on the microfluidic chip for 6 infectious diarrhea pathogen samples;
the reference numbers in the figures are:
11. a reaction detection zone; 12. a sample introduction pool; 13. a distribution pool; 14. an amplification pool; 15. capillary micro valves.
A to H: a reaction detection zone; 1-8: an amplification pool that coats the primer set.
Hereinafter, the amplification chamber 1 at the reaction detection area A is denoted by A-1, the amplification chamber 2 at the reaction detection area A is denoted by A-2, and so on.
Detailed Description
The invention provides a primer composition for detecting infectious diarrhea pathogens, a kit and a detection method thereof, wherein the infectious diarrhea pathogens comprise at least one of rotavirus, adenovirus in intestines, norovirus, salmonella, shigella and campylobacter jejuni, and the primer composition comprises at least one group of primer groups corresponding to the infectious diarrhea pathogens.
Rotavirus, enterovirus, norovirus, enterovirus EV71, enterovirus CA16 and adenovirus type 3 adopted by the invention are clinical samples and are taken from a pediatric hospital affiliated to Shanghai Compound denier university; the salmonella is from China medical bacteria preservation management center (CMCC), and the strain number is CMCC 50115; the shigella is from China medical bacteria Collection management center (CMCC), and the strain number is CMCC 51592; campylobacter jejuni is from China center for culture Collection of Industrial microorganisms (CICC), with strain number CICC 22936; vibrio cholerae is from the American Type Culture Collection (ATCC) under strain number ATCC 27070; the Vibrio alginolyticus is from China center for culture Collection of Industrial microorganisms (CICC), and the strain number is CICC 21664; the Escherichia coli is from China medical bacteria preservation management center (CMCC), and the strain number is CMCC 44103; the internal control used in the present invention is a pseudovirus containing an amplified fragment of HBB interest.
The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example one
This example describes a microfluidic chip used in the present invention.
As shown in fig. 1, the microfluidic chip adopted in the present invention is a disc-shaped microfluidic chip, which includes 4 reaction detection areas 11, each reaction detection area 11 includes a sample injection pool 12, a distribution pool 13, a capillary micro valve 15 and an amplification pool 16, which are sequentially communicated, the sample injection pool 12 is communicated with the distribution pool 13 through an arc-shaped channel, a sample injection hole is arranged in the sample injection pool, and the distribution pool 13 is communicated with a waste liquid pool and an exhaust hole through an arc-shaped channel; each reaction detection zone 11 is provided with 8 amplification cells 16.
Wherein the sample injection pool 12 has the main function of loading reaction liquid; the distribution tank 13 is mainly used for uniformly distributing the reaction liquid to the amplification tank; the amplification pool 15 specifically realizes loop-mediated isothermal amplification (LAMP); the capillary micro valve 14 mainly utilizes the liquid retarding effect of the capillary to control the movement of the fluid in the micro-fluidic chip.
As shown in figures 2-3, the invention adopts 2 microfluidic chips to detect infectious diarrhea pathogens. A is a reaction detection area containing rotavirus nucleic acid; b is a reaction detection area containing the intestinal adenovirus nucleic acid; c is a reaction detection area containing norovirus nucleic acid; d is a reaction detection area containing salmonella nucleic acid; e is a reaction detection area containing shigella nucleic acid; f is a reaction detection area containing campylobacter jejuni nucleic acid; g is a reaction detection area containing six pathogen nucleic acids; h is a reaction detection area of the negative sample nucleic acid; respectively coating rotavirus primer groups, enterovirus primer groups, norovirus primer groups, salmonella primer groups, shigella primer groups, campylobacter jejuni primer groups and internal control primer groups in amplification pools 1, 2, 3, 4, 5, 6 and 8 in the reaction detection areas A-H; negative controls without RNase water were stored in the amplification cells 7 in the reaction detection zones A to F.
Example two
The present embodiment is a primer composition and a kit thereof for detecting infectious diarrhea pathogens, which are used in the present invention.
The primer composition comprises at least one of 6 independently packaged primer groups, wherein infectious diarrhea pathogens comprise at least one of six pathogens, namely rotavirus, enteroadenovirus, norovirus, salmonella, shigella and campylobacter jejuni, the 6 primer groups corresponding to the pathogens are a rotavirus primer group, an enteroadenovirus primer group, a norovirus primer group, a salmonella primer group, a shigella primer group and a campylobacter jejuni primer group respectively, the primer composition also comprises an internal control primer group, the internal control primer group is used for amplifying a human hemoglobin- β chain, and each primer sequence of each primer group is detailed in table 1.
The invention relates to a kit containing the primer composition, which further comprises a microfluidic chip coated with the primer group, isothermal amplification reaction liquid, isothermal amplification enzyme solution and a negative control substance. In the microfluidic chip coated with the primer groups, the molar ratio of the 4 primers in each primer group is as follows: primer with "F3" in 3. mu.M name, primer with "B3" in 3. mu.M name, primer with "FIP" in 12. mu.M name, and primer with "BIP" in 12. mu.M name. The volume of the isothermal amplification reaction solution in the kit was 25. mu.L, and contained 0.5. mu.L of 250mmol Tris-HCl, 0.5. mu.L of 300mmol KCl, 15. mu.L of 10mM dNTP, 0.5. mu.L of 120mM MgSO4, 0.25. mu.L of 1% (W/V) Tween-20, 0.5. mu.L of 50% (W/V) glycerol, 0.25. mu.L of 1% (W/V) PEG8000, and 1.0. mu.L of 10mM neutral red, and 25. mu.L was made up with RNase-free water. The isothermal amplification enzyme solution in the kit comprises 10 mu L of 10U/mu L DNA polymerase and 10 mu L of 20U/mu L reverse transcriptase solution.
The Bst DNA polymerase is a product of NEB company, Tris-HCl, KCl, MgSO4, Tween-20, glycerol, PEG8000 and neutral red are products of Sigma company, dNTP is a product of Takara company, and M-MLV reverse transcriptase is a product of Promega company.
EXAMPLE III
The present example is a method for detecting infectious diarrhea pathogens using the primer composition and the kit described in the second embodiment, which comprises the following steps:
1. coating of the primer composition;
as shown in fig. 2 and 3, a rotavirus primer group, an enterovirus primer group, a norovirus primer group, a salmonella primer group, a shigella primer group, a campylobacter jejuni primer group and an internal control primer group are respectively mixed with sucrose to prepare corresponding mixed solutions, and the final concentrations of each primer group and the sucrose in the mixed solutions are respectively 0.15 μ M and 1.0% (mass percentage); dropping 1 μ L of the mixed solution into corresponding amplification pools ( amplification pools 1, 2, 3, 4, 5, 6 and 8) of the microfluidic chip, storing RNase-free water in the amplification pool 7, drying the microfluidic chip in a 37 ℃ oven, tabletting, sealing a membrane, punching and vacuumizing, and coating a primer group in each amplification pool.
2. Extracting nucleic acid of a sample to be detected: extracting nucleic acid of a sample to be detected by adopting a magnetic bead method;
extracting rotavirus, enterovirus, norovirus, salmonella, shigella and campylobacter jejuni by using a nucleic acid extraction kit (Daan Gen., university of Zhongshan Co., Ltd.), and obtaining nucleic acid samples of the rotavirus, the enterovirus, the norovirus, the salmonella, the shigella and the campylobacter jejuni by performing the operation steps according to the nucleic acid extraction kit with a specification;
mixing the obtained rotavirus, enteroadenovirus, norovirus, salmonella, shigella and campylobacter jejuni nucleic acid according to the ratio of 1: 1: 1: 1: 1: 1, mixing in proportion to obtain multiple positive nucleic acid samples;
extracting enterovirus EV71, enterovirus CA16, adenovirus type 3, vibrio cholerae, vibrio alginolyticus and escherichia coli by using a nucleic acid extraction kit (Daan gene GmbH of Zhongshan university Co., Ltd.), and performing the operation steps according to the nucleic acid extraction kit with instructions, wherein the nucleic acids of the enterovirus EV71, the enterovirus CA16, the adenovirus type 3, the vibrio cholerae, the vibrio alginolyticus and the escherichia coli are expressed as 1: 1: 1: 1: 1: 1, mixing in proportion to obtain a negative nucleic acid sample;
extracting the internal control nucleic acid with a nucleic acid extraction kit (Daan Gen. GmbH, Zhongshan university);
the concentrations of the positive nucleic acid samples obtained above were all 103Copy/. mu.L, negative nucleic acid sample concentration of 106Copies/. mu.L, internal control nucleic acid concentration of 104TD/μL。
LAMP reaction
Mixing 25 mu L of constant temperature amplification reaction solution and 20 mu L of constant temperature amplification enzyme solution with 30 mu L of rotavirus nucleic acid and 10 mu L of internal control nucleic acid sample, and injecting into a sample injection pool of a reaction detection area A of the disc microfluidic chip;
mixing 25 mu L of constant-temperature amplification reaction solution and 20 mu L of constant-temperature amplification enzyme solution with 30 mu L of intestinal adenovirus nucleic acid and 10 mu L of internal control nucleic acid sample, and injecting into a sample injection pool of a reaction detection area B of the disc microfluidic chip;
mixing 25 mu L of constant-temperature amplification reaction solution and 20 mu L of constant-temperature amplification enzyme solution with 30 mu L of vibrio parahaemolyticus nucleic acid and 10 mu L of internal control nucleic acid sample, and injecting into a sample injection pool of a reaction detection area C of the disc microfluidic chip;
mixing 25 mu L of constant-temperature amplification reaction solution and 20 mu L of constant-temperature amplification enzyme solution with 30 mu L of salmonella nucleic acid and 10 mu L of internal control nucleic acid sample, and injecting into a sample injection pool of a reaction detection area D of the disc microfluidic chip;
mixing 25 mu L of constant-temperature amplification reaction solution and 20 mu L of constant-temperature amplification enzyme solution with 30 mu L of shigella nucleic acid and 10 mu L of internal control nucleic acid sample, and injecting into a sample injection pool of a reaction detection area E of the disc microfluidic chip;
mixing 25 mu L of constant-temperature amplification reaction solution and 20 mu L of constant-temperature amplification enzyme solution with 30 mu L of campylobacter jejuni nucleic acid and 10 mu L of internal control nucleic acid sample, and injecting into a sample injection pool of a reaction detection area F of the disc microfluidic chip;
mixing 25 mu L of constant-temperature amplification reaction solution and 20 mu L of constant-temperature amplification enzyme solution with 30 mu L of multiple positive nucleic acid and 10 mu L of internal control nucleic acid sample, and injecting into a sample injection pool of a reaction detection area G of the disc microfluidic chip;
mixing 25 mu L of constant-temperature amplification reaction solution and 20 mu L of constant-temperature amplification enzyme solution with 30 mu L of mixed negative sample nucleic acid and 10 mu L of internal control nucleic acid sample, and injecting the mixture into a sample injection pool of a reaction detection area H of the disc microfluidic chip;
sealing the sample injection pool of the microfluidic chip by a film, placing the sample injection pool in a centrifuge, and centrifuging at 3000rpm for 1 min; reacting for 10min in a constant temperature box at 50 ℃; reacting in a thermostat at 63 ℃ for 50 min.
4. Judging the result;
as shown in fig. 4 and 5, the LAMP reaction results were as follows: embedding a rotavirus primer group, and injecting rotavirus nucleic acid sample into amplification pools A-1 and G-1 which are pink, so that the two amplification pools A-1 and G-1 contain rotavirus nucleic acid and perform isothermal amplification reaction; the colors of the amplification pools B-2 and G-2, in which the enteric adenovirus primer groups are embedded and which are injected into the enteric adenovirus nucleic acid sample, are pink, which indicates that the two amplification pools B-2 and G-2 contain the enteric adenovirus nucleic acid and a constant-temperature amplification reaction occurs; the colors of the amplification pools C-3 and G-3 embedded with the norovirus primer group and injected with the norovirus nucleic acid sample are pink, which indicates that the two amplification pools C-3 and G-3 contain norovirus nucleic acid and isothermal amplification reaction occurs; the colors of the amplification pools D-4 and G-4 embedded with the salmonella primer group and injected with the salmonella nucleic acid sample are pink, which indicates that the two amplification pools D-4 and G-4 contain salmonella nucleic acid and a constant temperature amplification reaction occurs; the colors of the amplification pools E-5 and G-5 embedded with the Shigella primer group and injected with the Shigella nucleic acid sample are pink, which indicates that the two amplification pools E-5 and G-5 contain the Shigella nucleic acid and a constant-temperature amplification reaction occurs; the colors of the amplification pools F-6 and G-6 in which the campylobacter jejuni primer group is embedded and the nucleic acid sample of the campylobacter jejuni is injected are pink, which indicates that the two amplification pools F-6 and G-6 contain the nucleic acid of the campylobacter jejuni and the isothermal amplification reaction occurs; the amplification pools A-8, B-8, C-8, D-8, E-8, F-8, G-8 and H-8 which are embedded with the internal control primer group and injected with the internal control nucleic acid are pink, so that the eight amplification pools A-8, B-8, C-8, D-8, E-8, F-8, G-8 and H-8 contain the internal control nucleic acid and perform isothermal amplification reaction; and the other amplification pools are all light yellow, which indicates that the isothermal amplification reaction does not occur.
And related equipment can also be adopted for carrying out color interpretation in the microfluidic chip amplification pool.
The results show that the kit for detecting infectious diarrhea and the use method thereof can accurately detect the detection results of 6 pathogen indexes of rotavirus, enterovirus, norovirus, salmonella, shigella and campylobacter jejuni within 1 hour, and effectively improve the detection efficiency.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications or alterations to this practice will occur to those skilled in the art and are intended to be within the scope of this invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Shanghai Rapid diagnosis product Co., Ltd
<120> primer composition for detecting infectious diarrhea pathogens and kit thereof
<160>28
<170>PatentIn version 3.5
<210>1
<211>23
<212>DNA
<213>Artificial Sequence
<220>
<223> rotavirus primer F3
<400>1
cactatagat tggaaatcca gat 23
<210>2
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<223> rotavirus primer B3
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<210>3
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<213>Artificial Sequence
<220>
<223> rotavirus primer FIP
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actttccttg ccttaataac ccaatttgaa agacgatttg agtcg 45
<210>4
<211>47
<212>DNA
<213>Artificial Sequence
<220>
<223> rotavirus primer BIP
<400>4
ttcagaatgt tatttcgcaa caacatttga ttgtaaatca cgctcta 47
<210>5
<211>18
<212>DNA
<213>Artificial Sequence
<220>
<223> Enterovirus primer F3
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<212>DNA
<213>Artificial Sequence
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<223> Enterovirus primer B3
<400>6
tgtcaccacc attgtagcac 20
<210>7
<211>39
<212>DNA
<213>Artificial Sequence
<220>
<223> Enterovirus primer FIP
<400>7
aactaagggc gagggttgcg gtcagctcgt gtcgtgaga 39
<210>8
<211>40
<212>DNA
<213>Artificial Sequence
<220>
<223> Enterovirus primer BIP
<400>8
cagttgggca ctctaagggg acagggccat gaggacttga 40
<210>9
<211>20
<212>DNA
<213>Artificial Sequence
<220>
<223> norovirus primer F3
<400>9
gccctgacaa aactgaagga 20
<210>10
<211>18
<212>DNA
<213>Artificial Sequence
<220>
<223> norovirus primer B3
<400>10
cccccagtag ggacatca 18
<210>11
<211>42
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<213>Artificial Sequence
<220>
<223> norovirus primer FIP
<400>11
ttccaaacca accagctggg tcccccttgt catctccgaa ga 42
<210>12
<211>42
<212>DNA
<213>Artificial Sequence
<220>
<223> norovirus primer BIP
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ctggactagg ggtcccaacc atggtctttg ggagtgtgga at 42
<210>13
<211>20
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<223> Salmonella primer F3
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ggatgactcg ccatggtatg 20
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<213>Artificial Sequence
<220>
<223> Salmonella primer B3
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ttgttcaaca gctgcgtca 19
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<211>39
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<213>Artificial Sequence
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<223> Salmonella primer FIP
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ctgggcgaca agaccatcac catttgtcct ccgccctgt 39
<210>16
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<212>DNA
<213>Artificial Sequence
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<223> Salmonella primer BIP
<400>16
tccccgcatt gttgattgcg atccgcccca tattatccgt a 41
<210>17
<211>18
<212>DNA
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<223> Shigella primer F3
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<210>18
<211>19
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<220>
<223> Shigella primer B3
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<211>42
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<223> Shigella primer FIP
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<211>42
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<220>
<223> Shigella primer BIP
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acatgaagag cacgccaaca ccgcagagac ggtatcggaa ag 42
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<223> Campylobacter jejuni primer B3
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<223> campylobacter jejuni primer BIP
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<223> internal control primer F3
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<223> internal control primer FIP
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<220>
<223> internal control primer BIP
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gctgctggtg gtctaccctt gtcaggggtg gacagatcc 39
Claims (10)
1. A primer composition for detecting infectious diarrhea pathogens is characterized by comprising a rotavirus primer group, an enterovirus primer group, a norovirus primer group, a salmonella primer group, a shigella primer group and a campylobacter jejuni primer group;
wherein the rotavirus primer group comprises SEQ ID NO: 1, rotavirus primer F3 shown in SEQ ID NO: 2, rotavirus primer B3 shown in SEQ ID NO: 3 and the rotavirus primer FIP shown in SEQ ID NO: 4, rotavirus primer BIP;
wherein the intestinal adenovirus primer group comprises SEQ ID NO: 5, and the enteroadenovirus primer F3 shown in SEQ ID NO: 6, and the primers B3 of the enteroadenovirus shown in SEQ ID NO: 7 and the primers FIP and SEQ ID NO: 8, an enteroadenovirus primer BIP;
wherein the norovirus primer set comprises SEQ ID NO: 9, norovirus primer F3, SEQ ID NO: 10, norovirus primer B3, SEQ ID NO: 11 and norovirus primer FIP as shown in SEQ ID NO: 12, a norovirus primer BIP;
wherein the salmonella primer set comprises SEQ ID NO: 13, salmonella primer F3, SEQ ID NO: 14, salmonella primer B3, SEQ ID NO: 15 and the salmonella primer FIP shown in SEQ ID NO: 16, BIP as a salmonella primer;
wherein the Shigella primer set comprises SEQ ID NO: 17, shigella primer F3, SEQ ID NO: 18, shigella primer B3, SEQ ID NO: 19 and the shigella primer FIP shown in SEQ ID NO: 20, shigella primer BIP;
wherein the campylobacter jejuni primer group comprises SEQ ID NO: 21, and primers F3 and SEQ id no: 22, and a campylobacter jejuni primer B3 shown in SEQ ID NO: 23 and the primer FIP of campylobacter jejuni shown in SEQ ID NO: 24, and campylobacter jejuni primer BIP.
2. The primer composition for detecting infectious diarrhea pathogens according to claim 1, further comprising an internal control primer set for amplifying human hemoglobin- β chain;
wherein the internal control primer group comprises SEQ ID NO: 25, internal control primer F3 shown in SEQ ID NO: 26, internal control primer B3 shown in SEQ ID NO: 27 and the internal control primer FIP shown in SEQ ID NO: 28 as shown in figure 28.
3. A kit containing the primer composition of claim 1 or 2, further comprising a microfluidic chip, wherein the microfluidic chip is provided with 4 reaction detection areas which are not communicated with each other, each reaction detection area comprises a sample injection pool, a distribution pool, a capillary micro valve and an amplification pool which are sequentially communicated, each reaction detection area is provided with 8 amplification pools, and each primer component in the primer composition is respectively coated in the corresponding amplification pool.
4. The kit of claim 3, wherein in each amplification pool, the concentration of the primer F3 in each primer set is 1-4 μ M, the concentration of the primer B3 is 1-4 μ M, the concentration of the primer FIP is 8-15 μ M, and the concentration of the primer BIP is 8-15 μ M.
5. The kit according to claim 3, wherein the kit further comprises a constant temperature amplification reaction solution, a constant temperature amplification enzyme solution and a negative control.
6. The kit of claim 5, wherein the isothermal amplification reaction solution comprises 200-250 mM Tris-HCl, 300-400 mM KCl, 8-10 mM dNTP, 100-120 mM MgSO4, 1-5% (W/V) Tween-20, 40-50% (W/V) glycerol, 1-5% (W/V) PEG8000 and 10-20 mM indicator; the isothermal amplification enzyme solution comprises 10-12U/mu L of DNA polymerase and 20-30U/mu L of reverse transcriptase.
7. The kit of claim 6, wherein the indicator comprises HNB, Calcein, cresol red, phenol red, m-cresol purple, bromocresol purple, neutral red, naphtholphthalein, thymol blue.
8. The kit of claim 6, wherein the DNA polymerase is Bst DNA polymerase and the reverse transcriptase is M-MLV reverse transcriptase.
9. A method for detecting infectious diarrhea pathogens using the primer composition of claim 1 or 2 for non-diagnostic and therapeutic purposes, comprising the steps of:
step 1) coating of primer composition: coating each primer group in the primer composition in a corresponding amplification pool respectively, and fixing the primer group in the amplification pool through vacuum drying;
step 2) extracting nucleic acid of a sample to be detected: extracting nucleic acid of a sample to be detected by adopting a magnetic bead method;
step 3), LAMP reaction: mixing the constant temperature amplification reaction solution, DNA polymerase and reverse transcriptase solution with the nucleic acid of the sample to be detected, transferring to a sample injection pool of a micro-fluidic chip coated with a primer group, allowing the liquid to flow into the amplification pool under the action of centrifugal force, and then performing loop-mediated constant temperature amplification reaction;
step 4), interpretation of results: and (4) carrying out direct visual interpretation or adopting equipment to carry out color interpretation according to the color change in the amplification pool after the amplification is finished.
10. The method of claim 9, wherein the coating of the primer composition in step 1) comprises the following specific steps: mixing each primer group of the primer composition with sucrose to prepare a corresponding mixed solution, wherein the final concentrations of the primer group and the sucrose in the mixed solution are 0.15 mu M and 1.0 wt% respectively; dropping 1 μ L of the mixed solution into the corresponding amplification pool of the microfluidic chip, drying in a 37 ℃ oven, tabletting, sealing a membrane, punching and vacuumizing, and coating the primer group in the corresponding amplification pool.
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| CN107629951B (en) * | 2017-09-29 | 2021-03-02 | 深圳国际旅行卫生保健中心 | Micro-fluidic gene detection chip |
| CN108187769B (en) * | 2018-01-15 | 2020-08-28 | 西南石油大学 | Integrated type microscopic oil displacement chip capable of rotating a certain angle and being matched with mold |
| CN108531630A (en) * | 2018-06-06 | 2018-09-14 | 上海速创诊断产品有限公司 | The streptococcic primer sets of detection B races include the primer liquid and kit of the primer sets and its application |
| CN108754032B (en) * | 2018-07-19 | 2022-02-18 | 上海速创诊断产品有限公司 | Isothermal nucleic acid amplification system with high specificity and application thereof |
| CN110592250A (en) * | 2019-11-01 | 2019-12-20 | 中国人民解放军总医院 | LAMP primer combination for detecting intestinal pathogenic bacteria and application thereof |
| CN111057799A (en) * | 2019-11-22 | 2020-04-24 | 浙江大学 | Method for rapidly detecting pinnate mottle virus of sweet potato by using micro-fluidic chip and used primer |
| CN114606301A (en) * | 2020-12-07 | 2022-06-10 | 中国科学院脑科学与智能技术卓越创新中心 | Single cell transcriptome sequencing method |
| WO2024077197A1 (en) * | 2022-10-05 | 2024-04-11 | Life Technologies Corporation | Multiplex qpcr panel for gastrointestinal pathogens |
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