CN116064876A - Novel method for rapidly and accurately detecting infant klebsiella pneumoniae by combining polymerase chain reaction with nano fluorescent microsphere-immunochromatography technology - Google Patents
Novel method for rapidly and accurately detecting infant klebsiella pneumoniae by combining polymerase chain reaction with nano fluorescent microsphere-immunochromatography technology Download PDFInfo
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Abstract
The invention provides a novel method for rapidly and accurately detecting Klebsiella pneumoniae in infants by combining a polymerase chain reaction with a nano fluorescent microsphere-immunochromatography technology. The method comprises the following steps: designing and synthesizing an oligonucleotide primer by taking klebsiella pneumoniae specific target gene mdh as a target; extracting klebsiella pneumoniae genome DNA, and performing PCR amplification reaction by using the primers to obtain a sample amplification product; and purifying the PCR amplification product by using magnetic nano particles, and carrying out quick quantitative detection on the infant klebsiella pneumoniae by using a nano fluorescent microsphere immunochromatographic test strip. The invention uses the nano fluorescent microsphere material for a fluorescent lateral chromatography detection platform, combines signal amplification technologies such as polymerase chain reaction and the like, and greatly improves the sensitivity and the specificity of a detection system; the novel detection technology has strong applicability, can be used for rapidly and accurately detecting important pathogenic bacteria such as the Klebsiella pneumoniae of infants, meets the national actual demands, and has industrialization prospect.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and relates to a novel method for rapidly and accurately detecting Klebsiella pneumoniae in infants by combining Polymerase Chain Reaction (PCR) with a nano fluorescent microsphere-immunochromatography technology (FICTS).
Background
Klebsiella pneumoniae (Klebsiella pneumoniae) has become one of the most important pathogens for community-acquired and nosocomial infections in recent years, which can cause bacterial pneumonia, liver abscess, sepsis, neurological diseases, etc., as well as other life-threatening consequences. In clinical studies, drug-resistant klebsiella pneumoniae is found to be highly contagious, particularly in infants, because their immune system is very fragile. Infections caused by klebsiella pneumoniae are often accompanied by high mortality rates, which makes clinical anti-infective therapy a serious challenge.
At present, the traditional bacterial detection method based on a culture technology and a biochemical experiment is a conventional method for clinical detection at present, but the method has more steps, longer detection time and still needs to further improve the accuracy and the sensitivity; in addition, the operation level and experience of the detection technician also have an important influence on the accuracy of the detection result; the immunological method based on the antibody can shorten the detection time, but the quality and the specificity of the antibody can influence the detection result, and a plurality of pathogenic bacteria can not find the proper antibody, so that the method is limited in the scope and the range of clinical application. Therefore, the traditional pathogenic bacteria clinical detection technology is long in time consumption, and serious in missed detection, and cannot meet clinical diagnosis and treatment requirements. At present, the fluorescent quantitative PCR (RTFQ-PCR) method is widely applied due to the advantages of sensitivity, rapidness and quantification, and the gene chip, the sequencing, the MALDI-TOF MS technology, the SERS technology and the like, and has the advantages of high detection flux and high detection sensitivity, but the technology is expensive due to the matching equipment, the detection cost is increased, and the application of the technology in basic medical units such as village and town sanitariums and resource deficiency areas is limited. In addition, the lack of the current bioinformatics talents is faced with the sequencing result of massive information, so that the accurate and reliable analysis result is difficult to obtain in a short time, and the clinical popularization of the sequencing technology is limited.
The nucleic acid lateral flow immunochromatographic assay (Nucleic acid lateral flow immunoassay, NALFIA), also known as immunochromatographic strip technique (Immunochromatographic test strip, ICTS), is a solid-phase membrane immunoassay method combining an immunological technique, a chromatographic technique and an immunological labeling technique. Researchers use Quantum Dot Fluorescent Microspheres (QDFM) as tracers, and combine monoclonal antibodies of outer membrane proteins OMP28 and OMP22 of pathogenic bacteria Brucella onto nitrocellulose membranes respectively, so that ICTS test strips can be manufactured and early screening of Brucella can be performed. Ye et al developed two pairs of monoclonal antibodies by conventional hybridoma technology, one specific for the E.coli O157 antigen and the other pair specific for the E.coli H7 antigen. Two rapid detection kits were developed using these two pairs of antibodies to specifically detect the E.coli O157 antigen and E.coli H7 antigen, respectively. The colloidal gold test strip can be used for qualitatively detecting carbapenemase phenotype of carbapenem-resistant klebsiella pneumoniae (CRKP) and carrying out escherichia coli O157: h7 rapid detection with sensitivity up to 10 4 CUF/ml. Colloidal gold is a common marker for immunochromatography, and is widely applied at present, but the sensitivity of the colloidal gold is still a certain gap compared with other immunochromatography technologies, the colloidal gold cannot be used for accurate quantification, and most of colloidal gold is used for qualitative detection, so that the stability is still to be improved.
The fluorescent nanoparticle immunochromatography test strip (FICTS) can be successfully prepared by coating known antigens/antibodies and high-fluorescence-intensity and stable-performance Streptavidin-modified polystyrene nano fluorescent microspheres (strepitavidin-modified nanofluorescent polystyrene microspheres, nFM@SA) (shown in figure 1) on a microporous membrane solid-phase carrier, and can be used for rapid multifunctional detection of important pathogenic bacteria such as Klebsiella pneumoniae and the like. In the technology, the polystyrene nano fluorescent microsphere is used as a special nano material, has the advantages of good thermal stability, high fluorescence stability, high fidelity, high luminous efficiency and the like, can replace a colloidal gold material, can be used as an analysis marker to promote covalent connection of antibodies, and improves the sensitivity and the specificity of a detection technology. In the prepared test strip, nucleic acid amplification products (the products are modified with digoxin and biotin) are added to a sample adding area, a sample mixture to be tested passes through a membrane and reaches a detection line (T line) on a test strip through capillary action, at the moment, an antibody or an antigen on the test strip can be combined with an antigen or an antibody coated on the detection line, and a reaction result is quantitatively marked by taking fluorescent microspheres as markers (figure 4). In a word, the combination of the polymerase chain reaction and the nano fluorescent microsphere immunochromatography (PCR-FICTS) can be used as a rapid, cheap and quantitative detection method with strong specificity and high sensitivity, and provides a new tool for early diagnosis of diseases such as pneumonia.
Disclosure of Invention
Based on the defects of the prior art, the invention aims to provide a novel method for rapidly and accurately detecting the Klebsiella pneumoniae of an infant by combining a polymerase chain reaction technology with a nano fluorescent microsphere immunochromatography technology (PCR-FICTS).
The aim of the invention is achieved by the following technical means:
in one aspect, the invention provides a novel method for rapidly and accurately detecting Klebsiella pneumoniae in infants by combining a polymerase chain reaction with a nano fluorescent microsphere-immunochromatography technology, which comprises the following steps:
designing and synthesizing an oligonucleotide primer by taking klebsiella pneumoniae specific target gene mdh as a target;
extracting klebsiella pneumoniae genome DNA, and performing PCR amplification reaction by using the specific primers to obtain an amplification product;
purifying the sample product obtained by PCR amplification by using magnetic nanoparticles (FIG. 3);
and finally, carrying out quick quantitative detection on the infant klebsiella pneumoniae by adopting a nano microsphere immunochromatography test strip.
In the above method, preferably, the sequence of the primer is as follows:
an upstream primer: 5'-GCGTGGCGGTAGATCTAAGTCATA-3' (SEQ ID NO: 1)
A downstream primer: 5'-TTCAGCTCCGCCACAAAGGTA-3' (SEQ ID NO: 2).
The design and synthesis of the primer sequence are specifically as follows:
(1) Through experimental verification and literature retrieval, screening of klebsiella pneumoniae specific marker genes, especially sensitive specific marker genes;
(2) Primer 5.0, NCBI Primer-blast and other online design software (www.ncbi.nlm.nih.gov/tools/Primer-blast) are used for designing primers for the screened genes;
(3) And submitting the designed candidate sequence to GenBank for BLAST comparison, and screening out a sequence with better specificity for oligonucleotide primer synthesis.
In the above method, preferably, the reaction solution of the PCR amplification reaction includes:
in the above method, preferably, the reaction conditions of the PCR amplification reaction are as follows:
in the above method, the primers are labeled with digoxin and biotin, respectively, before PCR amplification.
In the above method, preferably, the specific step of purifying the sample product obtained by PCR amplification using the magnetic nanoparticle comprises:
(1) Taking 5 mu L of PCR amplified sample product in a sample tube, adding 9 mu L of PCR product treatment solution, gently mixing for 5s, standing at room temperature for combining for 5min, and reversing and mixing for 2 times;
(2) Then, the sample tube is placed on a magnetic rack for magnetic separation, and waste liquid is sucked and discarded;
(3) Then 200 mu L of 85% ethanol is slowly added into the sample tube, the sample tube is kept on the magnetic rack for 2min, the magnetic bead group is ensured to be adsorbed on the tube wall all the time, and the waste liquid is sucked and discarded;
(4) Repeating the step (3);
(5) Placing the sample tube at room temperature for 3-5 min until the ethanol is completely volatilized;
(6) Taking down the sample tube, adding 100 mu L of loading buffer solution, fully and uniformly mixing, standing at room temperature for 5min, and reversely and uniformly mixing for 2 times;
(7) And (3) placing the sample tube on a magnetic rack for magnetic separation, carefully sucking the supernatant into a new centrifuge tube, and preserving the obtained purified liquid for later use.
In the above method, preferably, the PCR product treatment solution is a silicon-coated magnetic nanoparticle high-salt suspension solution. The kit is purchased from Nanjing Tongna Biotechnology Co., ltd.
In the above method, preferably, the loading buffer is PBS (pH 7.4) buffer.
In the above method, preferably, the specific step of quantitatively detecting klebsiella pneumoniae genes by using an immunochromatographic test strip comprises the following steps:
(1) Taking out the immunochromatographic test strip card and making a mark;
(2) 100 mu L of loading buffer solution (PBS with pH of 7.4) is vertically added to a detection card to serve as a blank control, 100 mu L of treated PCR product purified solution is vertically dripped into a loading hole, and the solution is placed at room temperature for 2min;
(3) And (3) performing on-machine detection qualitative and quantitative analysis of the immunochromatographic test strip, and recording numerical values to realize qualitative and quantitative detection of klebsiella pneumoniae genes.
In the above method, preferably, the preparation of the immunochromatographic test strip card includes:
soaking the sample pad in PBS buffer solution with pH of 7.4, drying in a drying oven, and taking out for standby after 2 hours; spraying fluorescent microsphere-streptavidin solution onto NC membrane, anti-digoxin antibody onto detection line region (T line), biotin-bovine serum albumin onto quality control line region (C line) with spraying amount of 0.65uL/cm, and vacuum drying at 37deg.C overnight; and then sequentially sticking a sample pad, an NC film, an absorption pad and the like on a PVC bottom plate, cutting the sample pad, the NC film, the absorption pad and the like into test strips with the width of 4mm after the sample pad, the NC film, the absorption pad and the like are assembled, clamping the test strips, putting the test strips into an aluminum foil bag, adding a drying agent, sealing and preserving the test strips for later use.
In the method, the immunochromatography test strip detection principle is as follows:
ICTS contains 6 parts: sample pad, NC membrane, conjugate pad, detection and control lines, background pad, and absorbent pad. After PCR is loaded into a test strip loading hole (sample pad), the labeled PCR amplicon is combined with nano fluorescent microsphere-streptavidin (nFM-SA) on the test strip through biotin labeled at one end of the PCR amplicon; digoxin labeled through its other end binds to the anti-digoxin antibody in the T-line region (detection line region). Biotin-bovine serum albumin was immobilized on NC membrane as the C-line region (control line region).
In the above method, preferably, performing on-machine detection qualitative and quantitative analysis of the immunochromatographic test strip includes:
qualitative analysis: qualitative analysis is carried out by using a nucleic acid fluorescence quantitative analyzer, wherein a T value of more than 500 indicates that the target gene exists in the detection sample (the content of the target gene is judged according to the size of the T value), and a T value of less than 500 indicates that the target gene does not exist in the detection sample;
quantitative analysis: and (3) measuring the fluorescence values of the C value and the T value by using a nucleic acid fluorescence quantitative analyzer, drawing a standard curve by taking different DNA concentrations (or klebsiella pneumoniae concentrations) of klebsiella pneumoniae as an abscissa and taking the T/C value as an ordinate, and determining the number of the klebsiella pneumoniae in a common sample.
The invention has the beneficial effects that:
the novel method for rapidly and accurately detecting the infant klebsiella pneumoniae by combining the PCR with the nano fluorescent microsphere-immunochromatography technology creatively carries out multi-disciplinary cross research, uses the nano fluorescent microsphere material for a fluorescent lateral chromatography detection platform, combines with signal amplification technologies such as polymerase chain amplification reaction and the like, and greatly improves the sensitivity and the specificity of a detection system; the new detection technology has strong applicability, foolproof operation, low cost and easy popularization, can be used for rapidly and accurately detecting important pathogenic bacteria such as the Klebsiella pneumoniae of infants, meets the national actual demands, and has industrialization prospect; the magnetic beads are combined with the immunochromatography test strip, POCT (point of care testing) is rapidly detected on the purified PCR product, the specificity, sensitivity and detection efficiency of the detection technology are further improved on the basis of removing detection false positives, the manufactured portable POCT product is low in cost, small and easy to carry, the detection equipment is low in cost and small and easy to carry, the PCR product can be applied to basic medical and health units such as village and town sanitariums, and the like, the defects of methods such as clinical traditional detection technology, gene chips, fluorescent quantitative PCR, mass spectrum and Raman spectrum can be overcome, and a new scheme is provided for clinical accurate diagnosis and treatment. Compared with the prior art, the method has the following advantages:
(1) The amplified PCR product can be quantitatively detected by ICTS;
(2) The PCR product purified by the nano magnetic beads can eliminate background signals;
(3) The biotin-streptavidin system improves the sensitivity of the immunological binding and tracer assay;
(4) Compared with colloidal gold, the fluorescent microsphere has higher signal value, wider linear range and better batch-to-batch repeatability, and is suitable for quantitative detection;
(5) The obtained result is easy to read, and the matched instrument is small and light, thereby being beneficial to detecting the nucleic acid amplification product.
Drawings
FIG. 1 is a material characterization of nFM and nFM-SA. (A) is a Transmission Electron Microscope (TEM) photograph. And (B) is an ultraviolet-visible absorption spectrum test result. (C) is the fluorescence emission spectrum test result.
FIG. 2 is a flow chart of a PCR-FICTS detection technique.
FIG. 3 is a flow chart of the magnetic bead purification of PCR products.
Fig. 4 is a diagram of FICTS test strips and internal structures thereof.
In FIG. 5, (A) is the result of PCR-agarose gel electrophoresis sensitivity detection of Klebsiella pneumoniae (mdh) using serially diluted Klebsiella pneumoniae DNA samples: lane M,1200bp marker; lanes 1-8, DNA concentrations 7.7-7.7X10 -7 ng/. Mu.L, lane 9, negative control; the mdh gene product size is 346bp; (B) Is the lungCorrelation analysis of Klebsiella inflammatory DNA concentration and PCR-ICTS Signal value { Y= 30008.6-29388/(1+exp [ (x+1.3)/0.3 ]]) }. (C) Specific detection for PCR-agarose gel electrophoresis (Klebsiella pneumoniae and Klebsiella pneumoniae sample): lane M,1200bp marker; lane 1, klebsiella pneumoniae standard sample; lane 2, klebsite of pneumonia clinical samples; lanes 3-16, samples of non-klebsiella pneumoniae; lane 17, negative control.
In FIG. 6, (A) is a standard curve of RTFQ-PCR for Klebsiella pneumoniae mdh gene detection, and the ordinate represents CT (Cycle threshold) values; the abscissa is the quantitative Log concentration, i.e. the logarithm of the different concentration gradients;
(B) For detecting Klebsiella pneumoniae mdh by RTFQ-PCR method, 1-5 are respectively amplification curves corresponding to different concentration gradient dilution samples.
Detailed Description
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention. The starting materials or methods in the examples below are commercially available starting materials or conventional methods as are conventional in the art unless specifically indicated.
Example 1: PCR-ICTS detection
The embodiment provides a novel method for rapidly and accurately detecting Klebsiella pneumoniae genes in infants by combining PCR with a nano fluorescent microsphere-immunochromatography technology, which comprises the following steps:
(1) Extracting Klebsiella pneumoniae DNA, and measuring the OD value and concentration thereof;
(2) Carrying out PCR reaction, and preparing a PCR reaction liquid according to the following system:
in the above method, preferably, the reaction conditions of the PCR amplification reaction are as follows:
the primers used for the PCR are shown in Table 1:
TABLE 1 screening of Klebsiella pneumoniae specific Gene and primer design results (mdh Gene)
(3) Purification of PCR products
The detection flow chart is shown in fig. 3, and the operation steps are as follows:
(a) Taking 5 mu L of PCR amplification product in a sample tube, adding 9 mu L of PCR product treatment solution (silicon-coated magnetic nanoparticle high-salt suspension solution) into the sample tube, gently mixing the mixture for 5s, standing the mixture at room temperature for combining for 5min, and reversing the mixing process for 2 times;
(b) Placing the sample tube on a magnetic rack for magnetic separation, and sucking waste liquid;
(c) Slowly adding 200 mu L of 85% ethanol into the sample tube, keeping the sample tube on the magnetic rack for 2min, ensuring that the magnetic bead clusters are always adsorbed on the tube wall, and absorbing and discarding the waste liquid;
(d) Repeating step (c);
(e) The sample tube is placed at room temperature for 3 to 5 minutes until the ethanol is completely volatilized (grey brown magnetic beads, no liquid wall hanging exists);
(f) Taking down the sample tube, adding 100 mu L of loading buffer (PBS buffer with pH of 7.4), fully and uniformly mixing, standing at room temperature for 5min, and reversing and uniformly mixing for 2 times;
(g) And (3) placing the sample tube on a magnetic rack for magnetic separation, carefully sucking the supernatant into a new centrifuge tube, and preserving under proper conditions.
The comparison of the PCR products before and after purification is shown in Table 2.
Table 2: fluorescence intensity detection result of PCR-ICTS method before and after magnetic bead purification
Note that: p1, P2, P3 represent 3 PCR amplification products of the mdh gene.
As can be seen from table 2, the background signal (diluent) has lower fluorescence intensity, which indicates that the test strip itself does not generate false positive; the signal intensity after negative control purification was reduced to 187, indicating that the purification process removed most of the non-target bands; the signal values before and after purification of the groups P1, P2 and P3 are not obviously different, which shows that the purification process has extremely strong capability of screening the target strips.
(4) Immunochromatography test strip (ICTS) detection
The ICTS test strip and its internal structure are shown in FIG. 4. The preparation of the immunochromatography test strip card comprises the following steps:
soaking the sample pad in PBS buffer solution with pH of 7.4, drying in a drying oven, and taking out for standby after 2 hours; spraying fluorescent microsphere-streptavidin solution onto NC membrane, anti-digoxin antibody onto detection line region (T line), biotin-bovine serum albumin onto quality control line region (C line) with spraying amount of 0.65uL/cm, and vacuum drying at 37deg.C overnight; and sequentially sticking a sample pad, an NC film, an absorption pad and the like on a PVC bottom plate, cutting the sample pad, the NC film, the absorption pad and the like into test strips with the width of 4mm after the sample pad, the NC film, the absorption pad and the like are assembled, clamping the test strips, putting the test strips into an aluminum foil bag, adding a drying agent, sealing and preserving the test strips for later use.
The specific method for detecting the immunochromatographic test strip (ICTS) comprises the following steps:
(a) Taking out the immunochromatographic test strip card and making a mark;
(b) Vertically adding 100 mu L of loading buffer solution to a detection card to make blank control, vertically dripping 100 mu L of treated PCR product to a loading hole, and standing at room temperature for 2min;
(c) Performing on-machine qualitative and quantitative detection analysis of the immunochromatographic test strip, and recording the numerical value;
qualitative analysis: qualitative analysis is carried out by using a nucleic acid fluorescence quantitative analyzer, wherein a T value of more than 500 indicates that the target gene exists in the detection sample (the content of the target gene is judged according to the size of the T value), and a T value of less than 500 indicates that the target gene does not exist in the detection sample;
quantitative analysis: and (3) measuring the fluorescence values of the C value and the T value by using a nucleic acid fluorescence quantitative analyzer, drawing a standard curve by taking different DNA concentrations (or klebsiella pneumoniae concentrations) of klebsiella pneumoniae as an abscissa and taking the T/C value as an ordinate, and determining the number of the klebsiella pneumoniae in a common sample.
Comparative example 1: PCR-agarose gel electrophoresis detection of mdh gene of klebsiella pneumoniae
The comparative example provides a method for detecting mdh gene of klebsiella pneumoniae by adopting PCR-agarose gel electrophoresis, which specifically comprises the following steps:
(1) Extracting Klebsiella pneumoniae DNA, measuring the OD value and concentration of the Klebsiella pneumoniae DNA, and carrying out PCR reaction;
(2) Electrophoresis: 1.5% of gel was prepared, and data were read under electrophoresis conditions of 140V for 40min with a loading of 4. Mu.L.
Comparative example 2: fluorescent quantitative PCR (RTFQ-PCR) detection of mdh gene of klebsiella pneumoniae
The comparative example provides a method for detecting mdh gene of klebsiella pneumoniae by adopting fluorescent quantitative PCR (RTFQ-PCR), which specifically comprises the following steps:
(1) The reaction solution for PCR was prepared as follows:
(2) The reaction conditions for PCR were as follows:
pre-denaturation: 95 ℃ for 3min
Amplification: 95 ℃ for 15s;60 ℃ for 30s;72 ℃ for 30s
Cycle number: 45
(3) The standard curve of mdh gene was first detected with klebsiella pneumoniae standard bacteria (fig. 6), and then mdh detection was performed on clinical sample strains, and the detection results are shown in tables 3 and 4.
Table 3: PCR-ICTS (example 1) and PCR-gel electrophoresis (comparative example 1), and detection results (mdh gene) by clinical conventional detection methods
Note that: klebsiella pneumoniae (standard) is a Klebsiella pneumoniae standard strain; klebsiella pneumoniae for infants 1 -24 (clinical): a sample of collected clinical infant klebsiella pneumoniae.
The clinical traditional detection method comprises the following steps: the strain is identified by a microorganism culture method and a traditional biochemical identification method (a full-automatic bacteria identification instrument) combined with a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS).
Table 4: comparison of PCR-ICTS technology (example 1) with other methods of detection of Klebsiella pneumoniae
Note that: 1 comprising the following steps: extracting DNA, amplifying by PCR, and performing agarose gel electrophoresis; 2 comprising the following steps: DNA extraction, PCR amplification, magnetic bead purification and ICTS; 3 comprising the following steps: DNA extraction, RTFQ-PCR.
As can be seen from FIGS. 5 and 6, the PCR-FICTS technology has strong specificity, and the detection time is basically consistent with RTFQ-PCR; the minimum detection limit of the PCR-FICTS technology can reach 7.7X10 -6 ng/. Mu.L, RTFQ-PCR minimum detection limit of 2.5X10 -6 The sensitivity of the PCR-FICTS technology is basically consistent with that of RTFQ-PCR, but is higher than that of the traditional microorganism detection method and the PCR-agarose gel electrophoresis detection technology, and the detection cost of the PCR-FICTS technology is lower than that of the RTFQ-PCR.
As can be seen from Table 3, the PCR-FICTS and gel electrophoresis were used to detect 24 samples of clinical Klebsiella pneumoniae and 14 samples of non-Klebsiella pneumoniae, respectively, and the detection results prove that the technique can successfully detect the samples of clinical Klebsiella pneumoniae in infants.
The above examples describe in detail preferred embodiments of the present invention, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. A novel method for rapidly and accurately detecting infant klebsiella pneumoniae by combining polymerase chain reaction with nano fluorescent microsphere-immunochromatography (PCR-FICTS), which comprises the following steps:
designing and synthesizing an oligonucleotide primer by taking klebsiella pneumoniae specific target gene mdh as a target;
extracting klebsiella pneumoniae genome DNA, and performing PCR amplification reaction by using the specific primers to obtain an amplification product;
purifying a sample product obtained by PCR amplification by adopting magnetic nano particles;
and finally, carrying out quick quantitative detection on the infant klebsiella pneumoniae by adopting a nano microsphere immunochromatography test strip.
2. The method of claim 1, wherein the primer sequence is as follows:
an upstream primer: 5'-GCGTGGCGGTAGATCTAAGTCATA-3' (SEQ ID NO: 1)
A downstream primer: 5'-TTCAGCTCCGCCACAAAGGTA-3' (SEQ ID NO: 2).
3. The method according to claim 1 or 2, wherein the reaction solution of the PCR amplification reaction comprises:
4. a method according to claim 3, wherein the reaction conditions of the PCR amplification reaction are as follows:
5. the method of claim 1, wherein the specific step of purifying the sample product obtained by PCR amplification using magnetic nanoparticles comprises:
(1) Taking 5 mu L of PCR amplified sample product in a sample tube, adding 9 mu L of PCR product treatment solution, gently mixing for 5s, standing at room temperature for combining for 5min, and reversing and mixing for 2 times;
(2) Then, the sample tube is placed on a magnetic rack for magnetic separation, and waste liquid is sucked and discarded;
(3) Then 200 mu L of 85% ethanol is slowly added into the sample tube, the sample tube is kept on the magnetic rack for 2min, the magnetic bead group is ensured to be adsorbed on the tube wall all the time, and the waste liquid is sucked and discarded;
(4) Repeating the step (3);
(5) Placing the sample tube at room temperature for 3-5 min until the ethanol is completely volatilized;
(6) Taking down the sample tube, adding 100 mu L of loading buffer solution, fully and uniformly mixing, standing at room temperature for 5min, and reversely and uniformly mixing for 2 times;
(7) And (3) placing the sample tube on a magnetic rack for magnetic separation, carefully sucking the supernatant into a new centrifuge tube, and preserving the obtained purified liquid for later use.
6. The method of claim 5, wherein the PCR product processing solution is a silicon coated magnetic nanoparticle high salt suspension solution.
7. The method of claim 5, wherein the loading buffer is a PBS buffer at pH 7.4.
8. The method of claim 1, wherein the specific step of quantitatively detecting klebsiella pneumoniae genes using an immunochromatographic test strip comprises:
(1) Taking out the immunochromatographic test strip card and making a mark;
(2) 100 mu L of loading buffer solution (PBS with pH of 7.4) is vertically added to a detection card to serve as a blank control, 100 mu L of treated PCR product purified solution is vertically dripped into a loading hole, and the solution is placed at room temperature for 2min;
(3) And (3) performing on-machine detection qualitative and quantitative analysis of the immunochromatographic test strip, and recording numerical values to realize qualitative and quantitative detection of klebsiella pneumoniae genes.
9. The method of claim 8, wherein the preparing of the immunochromatographic test strip card comprises:
soaking the sample pad in PBS buffer solution with pH of 7.4, drying in a drying oven, and taking out for standby after 2 hours; spraying fluorescent microsphere-streptavidin solution (figure 1) onto NC membrane, anti-digoxin antibody onto detection line region (T line), biotin-bovine serum albumin onto quality control line region (C line) with spraying amount of 0.65uL/cm, and vacuum drying at 37deg.C overnight; and sequentially sticking a sample pad, an NC film, an absorption pad and the like on a PVC bottom plate, cutting the sample pad, the NC film, the absorption pad and the like into test strips with the width of 4mm after the sample pad, the NC film, the absorption pad and the like are assembled, clamping the test strips, putting the test strips into an aluminum foil bag, adding a drying agent, sealing and preserving the test strips for later use.
10. The method of claim 8, wherein performing on-machine detection qualitative and quantitative analysis of the immunochromatographic test strip comprises:
qualitative analysis: qualitative analysis is carried out by using a nucleic acid fluorescence quantitative analyzer, wherein a T value of more than 500 indicates that the target gene exists in the detection sample (the content of the target gene is judged according to the size of the T value), and a T value of less than 500 indicates that the target gene does not exist in the detection sample;
quantitative analysis: and (3) measuring the fluorescence values of the C value and the T value by using a nucleic acid fluorescence quantitative analyzer, drawing a standard curve by taking different DNA concentrations (or klebsiella pneumoniae concentrations) of klebsiella pneumoniae as an abscissa and taking the T/C value as an ordinate, and determining the number of the klebsiella pneumoniae in a common sample.
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