CN117987578B - Microfluidic chip for simultaneously detecting pathogenic escherichia coli, salmonella and drug resistance genes and application thereof - Google Patents
Microfluidic chip for simultaneously detecting pathogenic escherichia coli, salmonella and drug resistance genes and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a microfluidic chip for simultaneously detecting pathogenic escherichia coli, salmonella and drug resistance genes and application thereof. The microfluidic chip can be used for simultaneously detecting drug resistance genes of enterotoxigenic escherichia coli, enteropathogenic escherichia coli, enterohemorrhagic escherichia coli, salmonella enteritidis, salmonella typhimurium, salmonella pullorum, salmonella gallinarum and 4 antibiotics of quinolones, aminoglycosides, tetracyclines and chloramphenicol, and has the advantages of simple and rapid operation, high flux, less reagent consumption, high accuracy, high sensitivity and good repeatability, and has important significance for rapid and accurate diagnosis of clinical animal epidemic diseases and epidemiological investigation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a microfluidic chip for simultaneously detecting pathogenic escherichia coli, salmonella and drug-resistant genes and application thereof.
Background
Coli is a microorganism widely existing in intestinal tracts of people and various animals, pathogenic escherichia coli can cause intestinal tract and parenteral infection, cause local or systemic infection of livestock and poultry, seriously influence the growth and development of livestock and poultry, and bring about huge economic loss. Most salmonella only causes infections in animals such as livestock and poultry, but part of the serotypes of salmonella cause severe infections in humans. Pathogenic escherichia coli and salmonella can infect human beings through contaminated livestock and poultry products, so that a series of diseases such as urinary tract infection, sepsis, food poisoning and the like are caused, and the harm is serious.
The application of antibiotics in livestock and poultry raising clinic has some effects on prevention and control of diseases, but due to long-term and unreasonable use of antibiotics, bacterial drug resistance is generated and spread, and more drug-resistant escherichia coli and salmonella strains appear, so that the curative effect of the antibiotics is reduced or even disabled, part of infectious diseases which can be cured once become uncontrollable, and meanwhile, the residues of the antibiotics in livestock and poultry bodies are aggravated, the quality safety and public health safety of livestock and poultry products are seriously threatened, and potential harm is brought to the sustainable development of livestock and poultry raising industry, human health and ecological safety.
The detection technology for pathogenic escherichia coli and salmonella at present mainly comprises bacterial separation culture, biochemical identification, immunological technology, molecular biological technology and the like. The traditional detection means is time-consuming and low in detection efficiency, has long bacterial separation and culture period and complex operation, and cannot meet the requirement of rapid detection; the immunological technology has low sensitivity and specificity, and is not suitable for detecting mixed infection of multiple pathogens; the PCR technology is widely applied because of the advantages of strong specificity, high sensitivity, simple operation, rapid detection and the like. In clinic, the PCR technology mainly aims at detecting single pathogen, and simultaneously detects few pathogens aiming at different pathogens, and due to the complex serotypes of pathogens such as escherichia coli, salmonella and the like, the current multiple PCR technology can only detect a limited number of genes simultaneously, and in a multiple PCR detection system, interference is easy to occur among different pathogens, so that the accuracy of a result is influenced. Therefore, in order to improve the capability of rapidly detecting pathogenic escherichia coli and salmonella, it is highly desirable to develop a more efficient, rapid and accurate detection means for simultaneously monitoring a plurality of pathogenic escherichia coli and salmonella, so as to rapidly and accurately perform differential diagnosis on the pathogens, thereby being beneficial to preventing and controlling animal epidemic diseases and having positive significance for the healthy development of animal husbandry.
At present, in the detection of bacterial drug resistance, the traditional drug sensitivity test has a certain limitation, and the method has the advantages of long time consumption, strong experience dependence and complicated operation due to the need of culturing and purifying bacteria, so that the method cannot completely meet the requirements of clinical diagnosis and treatment. Bacterial resistance to antimicrobial drugs is mostly generated by the presence or acquisition of drug-resistant genes, so detection of drug-resistant genes is of great importance for determination of drug-resistant bacteria, and genotypic detection plays an important role in epidemiological investigation of infection and transmission of drug-resistant bacteria, discovery and identification of new drug-resistant strains. If a certain drug resistance gene can be detected before the drug administration, the method is favorable for guiding the clinical reasonable drug administration and delaying the development of drug resistance. Because the bacterial drug resistance mechanism is quite complex and is caused by a plurality of drug resistance genes, if the drug resistance genes are detected respectively, the workload is huge, and along with the continuous increase of clinical multi-drug resistant bacteria, the detection of a plurality of drug resistance genes of a plurality of antibiotics is needed. Therefore, it is very necessary to change one-to-one detection mode, develop a one-time multi-element detection method, and realize quick and accurate detection of bacterial drug resistance.
The gene chip technology is to orderly arrange a large number of target gene fragments on a carrier such as a glass slide, to use a known nucleic acid sequence as a probe to hybridize with a complementary target nucleotide sequence, to detect fluorescent signals by a chip scanner, and to perform data analysis by computer software. The gene chip technology has the characteristics of integration, micromation, automation, rapidness, high flux and the like. Compared with the traditional PCR, the PCR technology and the microfluidic chip can be combined to obtain more uniform system temperature, thereby being beneficial to improving the amplification yield and enabling the detection to be more sensitive. The microfluidic chip integrates the whole detection process into one chip, realizes automation, has low consumption of samples and reagents and high reaction speed, can simultaneously complete the identification of various pathogenic bacteria and the detection of various drug-resistant genes of drug-resistant strains in a high-flux parallel detection mode, and provides a powerful tool for the identification of pathogenic bacteria and the analysis of drug-resistant genes.
At present, a plurality of detection methods are established for differential diagnosis and drug resistance of pathogenic escherichia coli and salmonella respectively, however, no application of a microfluidic chip detection method capable of simultaneously detecting a plurality of pathogenic escherichia coli, salmonella and drug resistance genes is clinically available.
Therefore, a microfluidic chip detection method aiming at various pathogenic escherichia coli, salmonella and drug-resistant genes is established, so that diagnosis efficiency and accuracy are further improved, a more effective detection way is provided for clinical animal epidemic disease diagnosis, epidemiological investigation and other works, and the method has important significance for improving quality safety and public health safety risk monitoring capability of livestock and poultry products.
Disclosure of Invention
In one aspect, the invention provides a set of primers comprising 25 sets of primer combinations;
primer set 1 consists of SEQ ID NO:1 and 2;
primer set 2 consists of SEQ ID NO:3 and 4;
Primer set 3 consists of SEQ ID NO:5 and 6;
primer set 4 consists of SEQ ID NO:7 and 8;
Primer set 5 consists of SEQ ID NO:9 and 10;
primer set 6 consists of SEQ ID NO:11 and 12;
primer set 7 consists of SEQ ID NO:13 and 14;
primer set 8 consists of SEQ ID NO:15 and 16;
primer set 9 consists of SEQ ID NO:17 and 18;
primer set 10 consists of SEQ ID NO:19 and 20;
primer set 11 consists of SEQ ID NO:21 and 22;
Primer set 12 consists of SEQ ID NO:23 and 24;
primer set 13 consists of SEQ ID NO:25 and 26;
primer set 14 consists of SEQ ID NO:27 and 28;
Primer set 15 consists of SEQ ID NO:29 and 30;
primer set 16 consists of SEQ ID NO:31 and 32;
primer set 17 consists of SEQ ID NO:33 and 34;
primer set 18 consists of SEQ ID NO:35 and 36;
primer set 19 consists of SEQ ID NO:37 and 38;
primer set 20 consists of SEQ ID NO:39 and 40;
primer set 21 consists of SEQ ID NO:41 and 42;
primer set 22 consists of SEQ ID NO:43 and 44;
Primer set 23 consists of SEQ ID NO:45 and 46;
Primer set 24 consists of SEQ ID NO:47 and 48;
Primer set 25 consists of SEQ ID NO:49 and 50.
In some embodiments, the forward primer 5' end of the set of primers further has a universal tag sequence. In some specific embodiments, the universal tag sequence is identical to a fluorescent-labeled universal sequence; more specifically, it is consistent with the common sequence of FAM fluorescent markers in the PCR reaction Master Mix.
In some embodiments, the invention provides the use of the set of primers, any one of the following a1-a 6:
a1. identifying 12 pathogen identification genes and 13 drug resistance genes;
a2. Preparing a product for identifying 12 pathogen identification genes and 13 drug resistance genes;
a3. Identifying whether the pathogen to be tested has one or more of 12 pathogen-identifying genes, or
Whether the pathogen to be detected has one or more of 13 drug resistance genes;
a4. preparing a product for identifying whether a pathogen to be tested has one or more of 12 pathogen identification genes, or
Whether the pathogen to be detected has one or more products of 13 drug resistance genes;
a5. Identifying whether the test sample is infected with at least one of the pathogens having one or more of the 12 pathogen identification genes, or
Whether the test sample is infected with at least one of the pathogens having one or more of the 13 drug resistance genes;
a6. preparing a product for identifying whether a test sample is infected with at least one of the pathogens having one or more of the 12 pathogen identification genes, or
Whether the test sample is infected with a product having at least one of the pathogens of one or more of the 13 drug resistance genes; the 12 pathogen identification genes are phoA, LTI, sta, eaeA, bfpB, stx, stx2, invA, spy, sdf I, speC and glgC; the 13 drug resistant genes are aac (6 ') -Ib-cr, oqxA, oqxB, qnrS, aadA1, aph (3') -IIa, aacC2, aacC4 and tetA, tetB, tetM, floR, cmlA.
In one aspect, the invention provides a kit containing the set of primers, wherein the kit is used for any one of the following b1-b 3:
b1. identifying 12 pathogen identification genes and 13 drug resistance genes;
b2. identifying whether the pathogen to be tested has one or more of 12 pathogen-identifying genes, or
Whether the pathogen to be detected has one or more of 13 drug resistance genes;
b3. identifying whether the test sample is infected with at least one of the pathogens having one or more of the 12 pathogen identification genes, or whether the test sample is infected with at least one of the pathogens having one or more of the 13 drug resistance genes
In some embodiments, the step of packaging each primer individually is included.
In one aspect, the invention provides a microfluidic chip having the set of primers immobilized thereon.
In some embodiments, the invention provides the use of the microfluidic chip, any one of the following a1-a 6:
a1. identifying 12 pathogen identification genes and 13 drug resistance genes;
a2. Preparing a product for identifying 12 pathogen identification genes and 13 drug resistance genes;
a3. Identifying whether the pathogen to be tested has one or more of 12 pathogen-identifying genes, or
Whether the pathogen to be detected has one or more of 13 drug resistance genes;
a4. preparing a product for identifying whether a pathogen to be tested has one or more of 12 pathogen identification genes, or
Whether the pathogen to be detected has one or more products of 13 drug resistance genes;
a5. Identifying whether the test sample is infected with at least one of the pathogens having one or more of the 12 pathogen identification genes, or
Whether the test sample is infected with at least one of the pathogens having one or more of the 13 drug resistance genes;
a6. preparing a product for identifying whether a test sample is infected with at least one of the pathogens having one or more of the 12 pathogen identification genes, or
Whether the test sample is infected with a product having at least one of the pathogens of one or more of the 13 drug resistance genes; the 12 pathogen identification genes are phoA, LTI, sta, eaeA, bfpB, stx, stx2, invA, spy, sdf I, speC and glgC; the 13 drug resistant genes are aac (6 ') -Ib-cr, oqxA, oqxB, qnrS, aadA1, aph (3') -IIa, aacC2, aacC4 and tetA, tetB, tetM, floR, cmlA.
In one aspect, the present invention provides a method of identifying whether a test sample is infected with at least one of a pathogen having one or more of 12 pathogen identification genes, or whether the test sample is infected with at least one of a pathogen having one or more of 13 drug resistance genes, comprising the steps of:
Contacting the nucleic acid of the sample to be tested with the set of primers, the kit or the microfluidic chip by taking the nucleic acid of the sample to be tested as a template, and performing PCR reaction to judge as follows:
If the amplified product of a certain primer or a certain reaction hole of the microfluidic chip detects a fluorescent signal, the sample to be tested is infected or suspected to be infected with the pathogen corresponding to the primer or the reaction hole, or the pathogen infected or suspected to be infected by the sample to be tested has the drug-resistant gene corresponding to the primer or the reaction hole.
The 12 pathogen identification genes are phoA, LTI, sta, eaeA, bfpB, stx, stx2, invA, spy, sdf I, speC and glgC; the 13 drug resistant genes are aac (6 ') -Ib-cr, oqxA, oqxB, qnrS, aadA1, aph (3') -IIa, aacC2, aacC4 and tetA, tetB, tetM, floR, cmlA.
In some embodiments, the methods are used for diagnosis and treatment of non-disease.
In one aspect, the present invention provides a test system for identifying whether a test sample is infected with at least one of a pathogen having one or more of 12 pathogen identification genes, or whether the test sample is infected with at least one of a pathogen having one or more of 13 drug resistance genes, the test system comprising the following components:
1) A detection means for 12 pathogen-identifying genes and 13 drug-resistant genes;
2) A data processing means;
3) A result output means;
The detection components of the 12 pathogen identification genes and the 13 drug resistance genes comprise the set of primers, the kit or the microfluidic chip, and the 12 pathogen identification genes are phoA, LTI, sta, eaeA, bfpB, stx, stx2, invA, spy, sdf I, speC and glgC; the 13 drug resistant genes are aac (6 ') -Ib-cr, oqxA, oqxB, qnrS, aadA1, aph (3') -IIa, aacC2, aacC4 and tetA, tetB, tetM, floR, cmlA.
In some embodiments, the data processing means is configured to: judging whether the sample to be tested is infected with at least one of the pathogens with one or more of the 12 pathogen identification genes or whether the sample to be tested is infected with at least one of the pathogens with one or more of the 13 drug resistance genes according to the detection of the 12 pathogen identification genes and the 13 drug resistance genes in the sample to be tested detected by the detection component.
The primer group comprises 12 pathogen identification genes of enterotoxigenic escherichia coli, enteropathogenic escherichia coli, enterohemorrhagic escherichia coli, salmonella enteritidis, salmonella typhimurium, salmonella pullorum and salmonella gallinarum and 13 drug resistance genes of 4 antibiotics such as quinolones, aminoglycosides, tetracyclines, chloramphenicol and the like. The invention also relates to a microfluidic chip detection method for simultaneously detecting 4 antibiotics drug resistance genes such as pathogenic escherichia coli, salmonella, quinolones, aminoglycosides, tetracyclines, chloramphenicol and the like by using the microfluidic chip primer group. The microfluidic chip detection method can be used for simultaneously detecting the drug resistance genes of enterotoxigenic escherichia coli, enteropathogenic escherichia coli, enterohemorrhagic escherichia coli, salmonella enteritidis, salmonella typhimurium, salmonella pullorum, salmonella gallinarum and 4 antibiotics of quinolones, aminoglycosides, tetracyclines and chloramphenicol, and has the advantages of simple and rapid operation, high flux, less reagent consumption, high accuracy, high sensitivity and good repeatability, and has important significance for rapid and accurate diagnosis of clinical animal epidemic diseases and epidemiological investigation.
The microfluidic chip established by the invention can simultaneously finish the identification and detection of 25 specific genes in one PCR amplification process, simultaneously identify and detect various pathogenic escherichia coli, salmonella and 4 antibiotics drug resistance genes such as quinolones, aminoglycosides, tetracyclines, chloramphenicol and the like, realize rapid and high-flux parallel detection, have the advantages of rapidness, specificity, sensitivity, stability, less reagent consumption and the like, are simple and convenient to operate, overcome the problems of single, time-consuming and labor-consuming existing detection modes, and improve the detection efficiency and accuracy.
Drawings
FIG. 1 is a graph showing the result of amplification of a pathogen-identifying gene in example 1, wherein: 1. coli ETEC 337272;2. coli EPEC 186733;3. coli EPEC 340977;4. coli EHEC 3411585;5. salmonella enteritidis DK6;6. salmonella typhimurium ATCC 14028;7. salmonella pullorum 526;8. salmonella typhi ATCC 9184.
FIG. 2 is a graph showing the result of amplification of drug-resistant gene in example 1, in which ：1. T-aac(6')-Ib-cr;2. T-oqxA;3. T-oqxB;4. T-qnrS;5. T-aadA1;6. T-aph(3')-IIa;7.T-aacC2;8. T-aacC4;9. T-tetA;10. T-tetB;11. T-tetM;12. T-floR;13. T-cmlA.
FIG. 3 is a graph showing the results of 12 pathogen identification gene-specific assays in example 2.
FIG. 4 is a graph showing the results of the sensitivity test of 25 genes in example 3.
FIG. 5 shows the results of the first repeat test in example 4.
FIG. 6 shows the results of the second repeatability test of example 4.
FIG. 7 shows the results of the third repeatability test of example 4.
FIG. 8 is a chip site layout diagram in example 5.
FIG. 9 is a graph of the results of a partial blind sample test in example 6.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.
As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
The term "comprising" is used in this specification or claims and is intended to include additional elements or steps in a manner similar to the term "comprising" as a transitional word in a claim. Furthermore, to the extent that the term "or" (e.g., a or B) is used, it is intended to refer to "a or B or both. When meaning "a or B only, but not both", then the term "a or B only, but not both" is employed. Thus, the use of the term "or" herein is intended to be inclusive, rather than exclusive, of the use. When the terms "and" or "are used together, as in" a and/or B ", this indicates a or B and a and B.
Universal experimental method
1. Material
1.1 Strain and drug-resistant gene reference
Strains: enterotoxigenic escherichia coli ETEC 337272, enteropathogenic escherichia coli EPEC 186733, enteropathogenic escherichia coli EPEC 340977, enterohemorrhagic escherichia coli EHEC 341158, salmonella enteritidis DK6, salmonella typhimurium ATCC 14028, salmonella pullorum 526, salmonella gallinarum ATCC 9184; drug resistance gene reference: the positive recombinant plasmid DNA of 13 drug resistance genes is constructed aiming at quinolone, aminoglycoside, tetracycline and chloramphenicol 4 antibiotics.
1.2 Reagent(s)
2X Master mix (universal label containing FAM mark) for chip detection is a product of Beijing Boao classical biotechnology limited company; the bacterial culture medium is a product of Beijing land bridge technology Limited liability company; the bacterial genome extraction kit is a product of Tiangen biochemical technology (Beijing) limited company.
1.3 Main instrument
The chip film coating instrument, the desk type multifunctional centrifugal machine PC16, the chip heat sealing instrument CHS1, the flat-plate PCR instrument and the chip scanner LuxScan-10K/D are all manufactured by Beijing Boao classical biotechnology limited company.
1.4 Microfluidic chip
The microfluidic chip is an IMAP chip, is a detection and analysis chip formed by 28×4 reaction holes, each row is formed by 28 reaction holes, and one chip can detect 4 samples simultaneously. The gene chip is a product of Beijing Boao classical biotechnology limited company. Each reaction hole detection system is 1 mu L.
The detection process comprises the following steps: designing and synthesizing a specific primer for detecting a microfluidic chip aiming at a target gene, pre-preparing the primer of the gene to be detected into a reaction hole, injecting a template DNA and a reaction Master Mix through a sample injection hole, injecting the DNA template and the reaction Master Mix into the reaction hole through centrifugation for amplification reaction, and finally detecting a fluorescent signal through a chip scanner.
2. Principle of experiment
A pair of specific primers is designed according to the specific DNA sequences of different target genes, a universal label sequence is added at the 5' end of the forward primer, and the universal label sequence is consistent with the universal sequence of FAM fluorescent markers in a PCR reaction Master Mix. In the PCR reaction process, the double-stranded general sequence is denatured into single strands to participate in the PCR reaction, so that FAM fluorescent groups and the Quencher are separated to generate fluorescent signals, and the fluorescent intensity is in linear relation with the number of target DNA molecules in a reaction system. The emitted signal and the intensity thereof can be detected by the chip scanner to obtain a signal value, and whether a specific sequence exists in the test sample is judged according to the signal value and the result of fluorescent scanning of the pseudo-color image.
Example 1 primer design and preparation of microfluidic chip
1. Primer design
Based on the gene sequences retrieved in GenBank, 12 pathogen identification gene primers for microfluidic chip detection against enterotoxigenic escherichia coli (ETEC), enteropathogenic escherichia coli (EPEC), enterohemorrhagic escherichia coli (EHEC), salmonella enteritidis, salmonella typhimurium, salmonella pullorum and salmonella gallinarum, and 13 drug resistance gene primers for microfluidic chip detection against quinolone, aminoglycoside, tetracycline and chloramphenicol 4-type antibiotic resistance genes were designed using molecular biology software, and primer specificity verification was performed using website BLAST tool. And adding a universal label sequence at the 5' end of the forward primer, wherein the universal label sequence is consistent with the universal sequence of FAM fluorescent markers in a PCR reaction Master mix. Primer sequence information is shown in Table 1.
Primer screening and verification result
Primers with better specificity (shown in table 1) are respectively screened for 12 pathogen identification genes and 13 drug resistance genes, and specific fluorescent signals are detected, and no non-specific signals are detected to be consistent with expectations. The results are shown in FIGS. 1 and 2.
2. Preparation of strains and positive references
2.1 Culture of strains
The bacterial isolation and culture were performed according to the conventional procedure.
2.2 Extraction of bacterial genomic DNA
Extracting genome DNA from bacterial cultures of standard strains of enterotoxigenic escherichia coli, enteropathogenic escherichia coli, enterohemorrhagic escherichia coli, salmonella enteritidis, salmonella typhimurium, salmonella pullorum, salmonella gallinarum and clinically isolated escherichia coli respectively, and extracting according to a bacterial genome extraction kit instruction.
2.3 Preparation of positive recombinant plasmid DNA of drug resistance gene
2.3.1 Strains and major reagents: the 13 strains of the escherichia coli are identified by biochemical and fluorescent PCR; the strain E.coli DH5 alpha is a product of Beijing Ding Guo prosperous biotechnology limited liability company; GV Topo-TA vector, product of Genview company; the DNA purification and recovery kit, the plasmid extraction and purification kit, ecoRI and EcoRV restriction enzymes are all manufactured by Promega company.
2.3.2 Drug resistance Gene amplification and cloning
According to the gene sequences searched in GenBank, 13 drug resistance gene primers aiming at quinolone, aminoglycoside, tetracycline and chloramphenicol 4 antibiotics are designed by using molecular biology software, and the specificity verification of the primers is carried out by using a website BLAST tool, and the related information of 13 drug resistance genes for cloning is shown in Table 2.
Extracting bacterial genome DNA, amplifying 13 drug-resistant genes by PCR, and then recovering and purifying DNA fragments. And connecting the recovered and purified PCR product to a GV Topo-TA vector, transforming the competent cells of the escherichia coli DH5 alpha, coating the transformed bacterial liquid on an LB plate containing ampicillin, cloning and detecting. Plaque PCR identification was performed using specific primers for 13 drug resistance genes, and plaques identified as positive clones by PCR were transferred to LB medium containing ampicillin, cultured overnight with shaking, and plasmid DNA was extracted. The plasmid was digested with EcoRI/EcoRV restriction enzymes, and recombinants were identified. Sequencing and analyzing after enzyme digestion identification is positive.
3. Prespotting of on-chip primers
The primers with the concentration of 1 mu M are respectively spotted into the holes of the chip according to the sequence of the table 1, each hole is spotted with 0.14 mu L, and after the primers are dried, the film is sealed by a film laminating instrument.
4. PCR reaction system and amplification program
The reaction system is as follows: 2x Master mix 0.5. Mu.L of template DNA was added and the total volume was 1. Mu.L.
The amplification procedure was: 95 ℃ for 15min;95 ℃ 20s,61-55 ℃ 60s,10 cycles (-0.6 ℃/cycle); 95 ℃ 20s,55 ℃ 60s,26 cycles; 60s at 10 ℃.
5. PCR amplification
Injecting the prepared reaction system into the chip from the chip inlet by using a liquid shifter, and putting the chip into a centrifugal machine to be centrifuged for 1min at 4000 rpm; placing the mixture into a chip heat sealing instrument for heat sealing for 1s; the mixture was put on a plate PCR instrument to carry out amplification reaction.
The reaction hole of the spotted saturated fluorescent dye is used as a positive quality control point, and the reaction hole of the primer and the fluorescent dye is used as a negative quality control point.
6. Data acquisition
After the PCR amplification program is finished, placing the microfluidic chip into a chip scanner to scan and detect the emitted signals and the intensities thereof, obtaining signal values, and judging whether a specific sequence exists in the test sample according to the signal values and the fluorescent scanning pseudo-color image result.
The positive judgment standard is as follows: the signal value is more than or equal to 1000, and no nonspecific signal value exists.
7. Sequencing verification of amplified products
And (3) carrying out micro-fluidic chip amplification on 25 genes, and then selecting PCR products of positive samples for clone sequencing. The result shows that the amplified products of the 25 gene microfluidic chips are completely consistent with the sequences corresponding to the target genes. The authenticity of the amplification result was confirmed.
Example 2 specificity experiments
The 12 pathogen identification genes were validated by a specificity test. For each gene, 1 target positive reaction bacteria, 1 negative control bacteria in genus, 5 bacteria in genus (Pasteurella multocida, proteus, klebsiella, staphylococcus aureus, pseudomonas aeruginosa) were selected, and 12 pathogen identification genes in the microfluidic chip prepared in example 1 were specifically verified. The results showed that the 12 pathogen identification genes did not cross-react with both the negative control bacteria in genus and the bacteria outside genus, and the specificity was good (FIG. 3, table 3).
And (3) carrying out conventional PCR detection verification and PCR product clone sequencing on a positive sample to prove that the 13 drug-resistant genes have no cross with other related drug-resistant genes and no nonspecific.
Example 3 sensitivity experiment
To verify the sensitivity of the 25 gene microfluidic chip detection method prepared in example 1, positive DNA concentrations extracted from standard strains corresponding to 12 pathogen identification genes and positive DNA concentrations extracted from clinically isolated escherichia coli corresponding to 13 drug-resistant genes were adjusted to 4 ng/μl for 25 target genes, 10-fold gradient dilutions were performed, and 10 dilutions were performed for each target gene, namely ：4 ng/µL、400 pg/µL、40 pg/µL、4 pg/µL、4×10-1pg/µL、4×10-2pg/µL、4×10-3pg/µL、4×10-4pg/µL、4×10-5pg/µL、4×10-6pg/µL. sensitivity tests were performed in microfluidic chips for 10 DNA concentrations.
The results show that the detection sensitivity of 25 target genes is between 40 pg/muL and 4×10 -3 pg/muL, which indicates that the method has higher sensitivity (fig. 4 and table 4).
Example 4 repeatability experiments
The 25 gene microfluidic chip prepared in example 1 was subjected to a repetitive amplification test 3 times and 3 replicates each time, 1 positive reaction bacteria, 1 negative control bacteria, and 3 NTC controls were set for each target gene. The repeated detection results are completely consistent, all 25 target genes show stable repeatability, and the repeated detection stability is good (fig. 5, 6 and 7).
Example 5 chip spotting
According to the results of examples 1-4, microfluidic chip design and site placement were performed.
Microfluidic chip design and site placement: each row had 28 wells, for a total of 4 rows. One chip can detect 4 samples simultaneously and 25 genes (12 pathogen identification genes and 13 drug resistance genes). The reaction wells numbered 1 and 27 served as positive control points, saturated fluorochromes were spotted, pathogen identification gene primers were spotted on the 2 nd to 13 th reaction wells, drug resistance gene primers were spotted on the 14 th to 26 th reaction wells, and the reaction well numbered 28 served as blank wells (negative control points), without primers and fluorochromes (fig. 8, table 5).
Example 6 evaluation of clinical application
And collecting clinical samples and performing application evaluation on the established method.
200 Samples are collected from the Beijing area Yanqing, the paraxial large-scale pig farm and chicken farm, and the separated 63 pathogenic bacteria are subjected to pathogen identification and drug resistance gene detection by adopting a microfluidic chip detection method, and partial results are shown in figure 9.
The pathogen identification result of the 63-strain pathogen microfluidic chip is completely consistent with the fluorescence PCR detection result. 2 methods all detect E.coli 53 strains, wherein E.coli (ETEC) 8 strains, E.coli (EPEC) 7 strains, E.coli (EHEC) 7 strains, and E.coli 31 strains; and detecting 10 strains of salmonella, wherein the salmonella enteritidis is 2 strains, the salmonella typhimurium is 1 strain, the salmonella pullorum is 3 strains and the salmonella gallinarum is 4 strains.
The detection result of the drug resistance gene of the 63-strain pathogenic bacteria microfluid chip is completely consistent with the detection result of the conventional PCR. 13 drug-resistant genes are detected from 63 pathogenic bacteria by 2 methods, and the positive detection rates of the drug-resistant genes are completely consistent. Positive detection rates of drug-resistant genes are respectively ：aac(6')-Ib-cr（41.3%）、oqxA（42.9%）、oqxB（39.7%）、qnrS（33.3%）、aadA1（69.8%）、aph(3')-IIa（17.5%）、aacC2（31.7%）、aacC4（90.5%）、tetA（66.7%）、tetB（30.2%）、tetM（9.5%）、floR（57.1%）、cmlA（50.8%）.
Therefore, the invention can simultaneously finish the identification detection of 25 specific genes in one reaction, has high sensitivity and specificity, overcomes the problems of single detection mode, time and labor waste in the prior art, realizes rapid and high-flux parallel detection, and has simple and convenient operation and less reagent consumption.
Claims (8)
1. A microfluidic chip, wherein a set of primers is immobilized on the chip, the set of primers comprising the following 25 sets of primer combinations:
primer set 1 consists of SEQ ID NO:1 and 2;
primer set 2 consists of SEQ ID NO:3 and 4;
Primer set 3 consists of SEQ ID NO:5 and 6;
primer set 4 consists of SEQ ID NO:7 and 8;
Primer set 5 consists of SEQ ID NO:9 and 10;
primer set 6 consists of SEQ ID NO:11 and 12;
primer set 7 consists of SEQ ID NO:13 and 14;
primer set 8 consists of SEQ ID NO:15 and 16;
primer set 9 consists of SEQ ID NO:17 and 18;
primer set 10 consists of SEQ ID NO:19 and 20;
primer set 11 consists of SEQ ID NO:21 and 22;
Primer set 12 consists of SEQ ID NO:23 and 24;
primer set 13 consists of SEQ ID NO:25 and 26;
primer set 14 consists of SEQ ID NO:27 and 28;
Primer set 15 consists of SEQ ID NO:29 and 30;
primer set 16 consists of SEQ ID NO:31 and 32;
primer set 17 consists of SEQ ID NO:33 and 34;
primer set 18 consists of SEQ ID NO:35 and 36;
primer set 19 consists of SEQ ID NO:37 and 38;
primer set 20 consists of SEQ ID NO:39 and 40;
primer set 21 consists of SEQ ID NO:41 and 42;
primer set 22 consists of SEQ ID NO:43 and 44;
Primer set 23 consists of SEQ ID NO:45 and 46;
Primer set 24 consists of SEQ ID NO:47 and 48;
Primer set 25 consists of SEQ ID NO:49 and 50.
2. The microfluidic chip of claim 1, wherein the 5' end of the forward primer in the set of primers further has a universal tag sequence.
3. A kit comprising the microfluidic chip of claim 1 or 2.
4. Use of a microfluidic chip according to claim 1 or 2 for non-disease diagnostic purposes, any one of the following a1-a 6:
a1. identifying 12 pathogen identification genes and 13 drug resistance genes;
a2. Preparing a kit for identifying 12 pathogen identification genes and 13 drug resistance genes;
a3. Identifying whether the pathogen to be tested has one or more of 12 pathogen-identifying genes, or
Whether the pathogen to be detected has one or more of 13 drug resistance genes;
a4. preparing a kit for identifying whether a pathogen to be tested has one or more of 12 pathogen identification genes, or
A kit for determining whether the pathogen to be tested has one or more of 13 drug-resistant genes;
a5. Identifying the presence or absence of a pathogen having one or more of 12 pathogen-identifying genes in the test sample, or
Whether pathogens with one or more of 13 drug resistance genes exist in the sample to be tested;
a6. preparing a kit for identifying the presence or absence of a pathogen having one or more of 12 pathogen identification genes in a test sample, or
A kit for detecting the presence of a pathogen having one or more of 13 drug-resistant genes in the sample;
the 12 pathogen identification genes are phoA, LTI, sta, eaeA, bfpB, stx, stx2, invA, spy, sdf I, speC and glgC; the 13 drug resistant genes are aac (6 ') -Ib-cr, oqxA, oqxB, qnrS, aadA1, aph (3') -IIa, aacC2, aacC4 and tetA, tetB, tetM, floR, cmlA.
5. Use of the kit of claim 3 for non-disease diagnostic purposes, any of the following b1-b 3:
b1. identifying 12 pathogen identification genes and 13 drug resistance genes;
b2. identifying whether the pathogen to be tested has one or more of 12 pathogen-identifying genes, or
Whether the pathogen to be detected has one or more of 13 drug resistance genes;
b3. identifying the presence or absence of a pathogen having one or more of 12 pathogen-identifying genes in the test sample, or
Whether pathogens with one or more of 13 drug resistance genes exist in the sample to be tested;
the 12 pathogen identification genes are phoA, LTI, sta, eaeA, bfpB, stx, stx2, invA, spy, sdf I, speC and glgC; the 13 drug resistant genes are aac (6 ') -Ib-cr, oqxA, oqxB, qnrS, aadA1, aph (3') -IIa, aacC2, aacC4 and tetA, tetB, tetM, floR, cmlA.
6. A method for identifying the presence or absence of a pathogen having one or more of 12 pathogen-identifying genes or one or more of 13 drug-resistant genes in a test sample for non-disease diagnostic purposes, comprising the steps of:
contacting the nucleic acid of the sample to be tested with 25 sets of primer combinations in the microfluidic chip according to claim 1 or 2 or 25 sets of primer combinations in the kit according to claim 3 by using the nucleic acid of the sample to be tested as a template, and performing the following evaluation after performing PCR reaction:
If the amplified product of one or more of the 25 sets of primer combinations detects a fluorescent signal, the presence or suspected presence of the primer combination in the sample to be tested corresponds to the detected pathogen, or
The pathogen has a drug resistance gene which is correspondingly detected by the primer combination;
the 12 pathogen identification genes are phoA, LTI, sta, eaeA, bfpB, stx, stx2, invA, spy, sdf I, speC and glgC; the 13 drug resistant genes are aac (6 ') -Ib-cr, oqxA, oqxB, qnrS, aadA1, aph (3') -IIa, aacC2, aacC4 and tetA, tetB, tetM, floR, cmlA.
7. A test system for identifying the presence or absence of a pathogen having one or more of 12 pathogen identification genes in a test sample or having one or more of 13 drug resistance genes in the test sample, the test system comprising:
1) A detection means for 12 pathogen-identifying genes and 13 drug-resistant genes;
2) A data processing means;
3) A result output means;
The detection means of the 12 pathogen identification genes and the 13 drug resistance genes comprise the microfluidic chip of claim 1 or 2, or the kit of claim 3;
the 12 pathogen identification genes are phoA, LTI, sta, eaeA, bfpB, stx, stx2, invA, spy, sdf I, speC and glgC; the 13 drug resistant genes are aac (6 ') -Ib-cr, oqxA, oqxB, qnrS, aadA1, aph (3') -IIa, aacC2, aacC4 and tetA, tetB, tetM, floR, cmlA.
8. The detection system of claim 7, the data processing means configured to:
Judging whether the pathogens with one or more of 12 pathogen identification genes exist in the sample to be detected or whether the pathogens with one or more of 13 drug resistance genes exist in the sample to be detected according to the detection of 12 pathogen identification genes and 13 drug resistance genes in the sample to be detected by the detection component.
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