CN110904249A - Nucleic acid detection kit and detection method for bacterial drug-resistant gene quantum dot chip - Google Patents
Nucleic acid detection kit and detection method for bacterial drug-resistant gene quantum dot chip Download PDFInfo
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Abstract
The invention discloses a nucleic acid detection kit and a detection method for gram-negative bacteria and gram-positive bacteria drug-resistant gene quantum dot chips. The kit comprises reaction liquid, a detection membrane strip and fluorescence detection liquid, wherein the reaction liquid comprises reaction liquid I, reaction liquid II and reaction liquid III; the fluorescence detection solution comprises quantum dots which are used for marking the surface of the capture probe and are coupled with streptavidin; the detection membrane strip comprises a nylon membrane and a capture probe fixed on the nylon membrane; the invention has the beneficial effects that: the kit and the method for detecting the nucleic acid of the bacterial drug-resistant gene quantum dot chip have the advantages of high flux, high sensitivity and high specificity, have fewer steps than the existing chromogenic gene chip, obviously shorten the detection time, have lower equipment cost than the organic fluorescent gene chip, and are favorable for clinical popularization.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a bacterial drug resistance gene quantum dot chip nucleic acid detection kit and a detection method, and particularly can be used for detecting drug resistance genes of gram-negative bacteria and gram-positive bacteria.
Background
Currently, with the use of a large amount of antibacterial drugs, a large amount of bacteria with Multiple Drug Resistance (MDR), extensive drug resistance (XDR) and full drug resistance (PDR) appear, the bacterial drug resistance tends to be multiple and has various forms and rapid change, and the speed of research and development of new drugs often lags behind the speed of generation of bacterial drug resistance, so that the rapid detection of a drug resistance mechanism has important significance.
The drug resistance mechanism of G-bacteria mainly comprises four mechanisms, namely modification or destruction of enzyme to antibiotics (mainly β -lactamase), membrane permeability change, active pumping mechanism and change or generation of new targets, wherein β -lactamase is a type of enzyme capable of specifically hydrolyzing β -lactam ring, is the most main mechanism for causing drug resistance of bacteria, especially G-bacteria, and accounts for 80% of various drug resistance mechanisms, β -lactamase is reported to be more than 200 so far, β -lactamase is mainly classified into four types (A/B/C/D), A mainly comprises TEM type, SHV type, CTX-M type (including four types), KPC and the like, B is also called as metallo β -lactamase and mainly comprises IMP type, VIM type, NDM type and the like, C mainly comprises DHA type and the like, and D mainly comprises OXA type and the like.
Liu et al first reported in China that plasmid-mediated mcr-1 genes render enterobacteriaceae of human and animal origin resistant to polymyxin, and subsequently reported in other countries worldwide that the mcr-1 gene was detected in enterobacteriaceae, including western European and American countries such as the United states, Latin America, Italy, the United kingdom, etc., global prevalence of "superbacteria" carrying mcr-1 genes, has pounded the alarm bell of enterobacteriaceae producing ultra-broad spectrum β lactamase or carbapenemase while being resistant to polymyxin.
M RSA is except to xixixixixulin drug resistance, to all β -lactam, macrolide, tetracycline, lincomycin, chloramphenicol, gentamicin, etc. can be drug resistant, MRSA locates in chromosome DN A inherent drug resistance, also have and locates in plasmid DN A acquired drug resistance, although its inherent drug resistance is relatively stable, compared with acquired drug resistance, its frequency of production is smaller, in clinical drug-resistant bacteria control in secondary position.
Vancomycin resistance gene: vancomycin is a glycopeptide antibacterial drug and acts by interfering with the synthesis of cell walls. There are five drug resistance genes of vancomycin: VanA, VanB, VanC, VanD and VanE types respectively have different drug-resistant gene cluster codes, wherein VanC is natural drug resistance, and the rest are acquired drug resistance. VanA is the most common, second-order vanab, of these several genotype species and is commonly found in enterococci and staphylococcus aureus.
The current methods for detecting bacterial drug resistance are as follows: the conventional drug sensitivity test, resistance protein test and the like can be used for judging the drug resistance of bacteria to antibacterial drugs, and the drug resistance can be obtained by measuring the diameter of a bacteriostatic circle, the MIC value and the IC value of a paper diffusion method, a broth dilution method and an E test according to NCCLS standard. The method for detecting the drug resistance gene of the antibacterial drug mainly comprises the following steps: PCR amplification, PCR-RFLP analysis, PCR-SSCP analysis, PCR-linear probe analysis, biochip technology, automatic DNA sequencing. The disadvantages of the methods are complicated operation, strong experience dependence and long report time.
Quantum Dots (QD), also known as semiconductor nanocrystals, are approximately spherical, have three-dimensional sizes in the range of 2-10nm, and have significant Quantum effects. The quantum dots are generally made of semiconductor materials of II-VI group elements (such as CdS, CdSe, CdTe, ZnSe, ZnS and the like) or III-V group elements (cadmium-free quantum dots, such as InP, InAs and the like), and a core/shell structure (such as common CdSe/ZnS core/shell structure quantum dots and the like) can also be made of two or more semiconductor materials. The physical, optical and electrical characteristics of the quantum dots are far superior to those of the existing organic fluorescent dye, and the quantum dots have the advantages of high sensitivity, good stability, long shelf life and the like, and are the best choice for a new generation of fluorescent labeled probe.
The quantum dot as a marking probe is particularly suitable for the application fields of high sensitivity, multi-index simultaneous detection and the like, and has the following advantages:
1) the quantum fluorescence efficiency is high, the molar extinction coefficient is large, the fluorescence intensity is more than 20 times stronger than the light intensity of the strongest organic fluorescent material, the quantum fluorescence detection device is suitable for high-sensitivity detection, and single quantum dot tracing can be realized by combining a high-resolution fluorescence microscope;
2) the optical-stability and photobleaching resistance are good, and the optical-bleaching resistant optical-film is suitable for long-time stable excitation dynamic observation and result archiving;
3) the fluorescence lifetime is long, the background fluorescence lifetime of the organic fluorescent dye or the biological sample is generally only 1-10 nanoseconds, the fluorescence lifetime of the quantum dots can last for 10-100 nanoseconds, and the background interference can be reduced and the sensitivity can be improved through the time resolution characteristic;
4) the emission wavelength is different due to the composition and the particle size, so that the quantum dots with similar characteristics but different emission wavelengths after surface modification are easy to prepare;
5) a broad and continuous absorption spectrum, realizing single light source multi-color excitation;
6) the emission spectrum is narrow and symmetrical, and the interference among different quantum dots in the multicolor excitation process can be reduced;
7) the quantum dots have larger Stokes shift and are easily distinguished from organic fluorescent dyes with smaller Stokes shift and background fluorescent light, and the background can be eliminated by adjusting the wavelength of the excitation light or using an optical filter, so that the sensitivity is improved.
8) The surface modified product has better biocompatibility, is coupled with various biomolecules, and has no non-specific adsorption.
Quantum dot materials were synthesized in glass matrix by Alexey I.Ekimov and in colloidal solution by Louis E.Brus in the 80 s of the 20 th century, and then the chemical modification technology of quantum dot surface ligands was gradually improved. As quantum dots have a plurality of advantages compared with the traditional fluorescent dyes, in 1998 Alivisatos and Nie, the quantum dots are applied to the biomolecular markers, and bioactive molecules such as antibodies or antigens are connected to active groups of quantum dot surface modifying bodies, so that the quantum dot bioluminescence dyeing is realized, and the application research of quantum dot biomarker materials is initiated.
The gene chip technology can simultaneously fix a large number of probes on a support to form a microarray, so that a large number of sequences in a clinical sample can be detected and analyzed at one time, and the defects of complex operation, low automation degree, small number of operation sequences, low detection efficiency and the like of the traditional nucleic acid Blotting hybridization (southern Blotting, Northern Blotting and the like) technology are overcome. The gene chip technology has the detection characteristics of high flux, high speed and high efficiency, so that a powerful detection tool is provided for the detection and identification of the pathogenic bacteria with high flux. However, the existing gene chip mainly uses organic fluorescent dye, BCIP/NBT, DAB and organic fluorescence as detection methods, and the organic fluorescent dye gene chip has many defects: the preparation process is complex, the detection process is complex and tedious, and the detection instrument needs a laser scanner with high cost, which is not favorable for clinical popularization. The BCIP/NBT and DAB modes have the technical defects of complicated operation steps, low detection sensitivity, poor repeatability and the like.
With the development of the technology, more researches are biased to molecular diagnosis at present, the types and the drug resistance conditions of pathogenic bacteria can be rapidly and accurately detected by utilizing the molecular technology, and the treatment efficiency is greatly improved. At present, the related technologies at home and abroad mainly comprise gene chips, a multiplex PCR technology, a fluorescent quantitative PCR technology, PCR-reverse dot hybridization and the like. However, the current real-time fluorescence quantitative PCR instrument has the problem of limited flux and cannot perform high-flux detection.
Therefore, the rapid detection of the pathogenic bacteria to determine the drug resistance of the pathogenic bacteria has important value in guiding the clinical reasonable selection of antibacterial drugs, provides basis for preventing and controlling the infection and the propagation of drug-resistant bacteria, provides a reliable technical means for monitoring and inspecting the drug-resistant epidemic situation, and has important practical significance.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a bacterial drug-resistant gene quantum dot chip nucleic acid detection kit with high flux, high sensitivity and high specificity and a detection method.
In order to solve the technical problems, the invention adopts the technical scheme that: the kit comprises a detection membrane strip, fluorescence detection liquid and reaction liquid, wherein the detection membrane strip comprises a nylon membrane and a capture probe fixed on the nylon membrane; the fluorescence detection solution comprises quantum dots which are used for marking the surface of the capture probe and are coupled with streptavidin.
The reaction solution comprises: reaction solution I, reaction solution II and reaction solution III.
The reaction solution I comprises the following primers:
primer VANAF: TGGCAAGTCAGGTGAAGATG (SEQ No. 1);
primer VANAR: CTAGACCTCTACAGCCGAGC (SEQ No. 2);
primer VANBF: CGTATTGACGTGGCTTTCCCG (SEQ No. 3);
primer VANBR: CGTGGATAGCGGCTGTACGAT (SEQ No. 4);
primer MEF GAAATGACTGAACGTCCG (SEQ No. 5);
primer MER: CATAACCTAATAGATGTGAAGTC (SEQ No. 6);
primer NDF: AGCCGCTGCATTGATGCTGAG (SEQ No. 7);
primer NDR: CGCGGTTGCTGGTTCGACC (SEQ No. 8);
primer CT9F: GCGTTGCAGTACAGCGACAATA (SEQ No. 9);
primer CT9R: ATATCATTGGTGGTGCCGTA (SEQ No. 10);
primer CT1F: GGACGATGTCACTGGCTGAGC (SEQ No. 11);
primer CT1R: CACAACCCAGGAAGCAGGCAGTC (SEQ No. 12);
primer MCF: GGCCTGCGTATTTTAAGCG (SEQ No. 13);
primer MCR: ATAGACACCGTTCTCACCCAG (SEQ No. 14);
primer VIMF: CGAGTGGTGAGTATCCGACAG (SEQ No. 15);
primer VIMR: GTCGTCATGAAAGTGCGTG (SEQ No. 16);
primer HBBF1: TATGGTTGGGATAAGGCTGG (SEQ No. 17);
primer HBBR1: CGAGCTTAGTGATACTTGTG (SEQ No. 18);
the reaction solution II contains the following primers:
primer O5F: GGCAACCACCACAGAAGTATT (SEQ No. 19);
primer O5R: CCAGCCTACTTGTGGGTCTAC (SEQ No. 20);
primer O2F: ATCTATATGGTAATGCTCTAAGC (SEQ No. 21);
primer O2R: ACCTCTTGAATAGGCGTAACCT (SEQ No. 22);
CAAAACAGCCGGTCACTG (SEQ No. 23);
primer AMR: AGCAACTGCTCATACGGCAT (SEQ No. 24);
primer KPF: AACTGACACTGGGCTCTGCACT (SEQ No. 25);
primer KPR: AATCCCTCGAGCGCGAGTCTA (SEQ No. 26);
GTAGCATTGCTACCGCAGCAG (SEQ No.27) as primer;
CACTACGTTATCTGGAGTGTGT (SEQ No. 28);
primer HBBF1: TATGGTTGGGATAAGGCTGG (SEQ No. 17);
primer HBBR1: CGAGCTTAGTGATACTTGTG (SEQ No. 18);
the reaction solution III is Hotstart Taq DNA polymerase.
Preferably, in the nucleic acid detection kit for bacterial drug-resistant gene quantum dot chip, the capture probe comprises:
probe VANAP: TCAATAGCGCGGACGAATT (SEQ No. 29);
probe VANBP: GTGACAAACCGGAGGCGAGGAC (SEQ No. 30);
GGCATCGTTCCAAAGAATGTA (SEQ ID NO: 31);
GCTGGGTCGAACCAGCAA (SEQ ID NO: 32);
probe CT9P: GTTGGTGACGTGGCTCAAA (SEQ No. 33);
probe CT1P: AGCCGACGTTAAACACCGCCA (SEQ No. 34);
TGAAGGTAATGAGCTTGCCAA (SEQ No.35) as a probe MCP;
ACACAGCGGCACTTCTCGC (SEQ ID NO: 36) as a probe VIMP;
probe O5P: AAACGCTTCCATTTAGCCC (SEQ No. 37);
probe O2P: CTCTAAGCCGCGCAAATAC (SEQ No. 38);
probe AMP CAGCAGTATCGGCCTGTTTGGTG (SEQ ID NO: 39);
probe KPP GTTGATTGGCTAAAGGGAAA (SEQ No. 40);
probe IMPP: CATTTAGCGGAGTTAACTAT (SEQ No. 41);
preferably, in the nucleic acid detection kit for bacterial drug-resistant gene quantum dot chip, the detection membrane strip further comprises an internal control probe for monitoring the extraction and amplification of sample nucleic acid: ICP1: TTTGCTAATCATGTTCATACC (SEQ No. 42).
Preferably, in the nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip, the capture probe is oligonucleotide single-stranded DNA, an amino group is labeled at the 3 'end or the 5' end of the oligonucleotide single-stranded DNA, an inter-arm is connected between the oligonucleotide single-stranded DNA and the amino group, the inter-arm is one or a combination of two of a fatty acid carbon chain and oligo dT (n), the fatty acid carbon chain is 1 to 12 carbon atoms in length, and n in the oligo dT (n) is an integer of 1 to 30.
Preferably, in the nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip, the 5' end of the reverse primer in the detection primer is modified with a biotin label, a spacer is connected between the detection primer and the biotin, the spacer is one or a combination of two of a fatty acid carbon chain and oligo dT (n), the length of the fatty acid carbon chain is 1-12 carbon atoms, and n in the oligo dT (n) is an integer of 1-30.
Preferably, in the above nucleic acid detection kit for a bacterial drug resistance gene quantum dot chip, the excitation wavelength of the quantum dot is 200-500nm, the emission wavelength of the quantum dot is 400-700nm, and the size of the quantum dot is 1-200 nm.
Preferably, in the nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip, the quantum dots are CdSe/ZnS core-shell structure quantum dots.
The invention has the beneficial effects that: the invention provides a nucleic acid detection kit for a bacterial drug-resistant gene quantum dot chip, which has high flux, high sensitivity and high specificity, establishes a bacterial drug-resistant gene quantum dot chip nucleic acid detection method for the first time and has the following advantages:
1) compared with the existing chromogenic gene chip, the kit has fewer detection steps, obviously shortens the detection time, has lower equipment cost (low light source requirement) than the organic fluorescent gene chip, has the carrier of the existing fluorescent gene chip which is glass, has complex preparation process and complex and fussy detection process, needs a laser scanner with high cost for a detection instrument, and is not beneficial to clinical popularization.
2) The invention adopts a multiple PCR method, the weight is up to 9, in one detection, the invention can simultaneously detect the common drug-resistant genes of 13 gram-negative bacteria and gram-positive bacteria, the amplification among the drug-resistant genes can not interfere with each other, and the specificity of the probe is high. The hybridization identification result by using the specific probe fixed on the hybridization membrane is faster and more accurate than that by using a culture method, and a certain basis is provided for clinical treatment. The invention can also guide the use of antibiotics, avoiding super-drug resistance caused by abuse of antibiotics.
3) The nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip can directly carry out related detection on clinical samples, the traditional drug sensitive detection needs to carry out separation, culture and biochemical reaction identification on bacteria in the samples, and the drug sensitive detection needs at least 3-4 days.
4) The nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip has high sensitivity and specificity, and all detection targets can reach 100copies/ul genome.
5) The invention is used for the detection kit of the bacterial drug-resistant gene quantum dot chip nucleic acid, and simultaneously, the detection of an internal control target is added, the internal control target is endogenous nucleic acid (human genome gene) of a sample, each sample can obtain an effective signal during detection, the whole-process monitoring of nucleic acid extraction to PCR amplification and quantum dot nucleic acid detection of each sample is ensured, and the occurrence of false negative is effectively avoided.
6) The nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip can use a quantum dot full-automatic nucleic acid hybridization instrument to detect optical signals, avoids errors caused by artificial interpretation to the maximum extent, and can realize high-throughput detection.
Drawings
FIG. 1 is a schematic diagram showing the detection results of the detection sensitivity of hybridization of each drug-resistant gene in the reaction solution I of the kit of the present invention according to the embodiment of the present invention.
FIG. 2 is a diagram showing the detection results of the detection sensitivity of hybridization of each drug-resistant gene in the reaction solution II of the kit of the present invention according to the embodiment of the present invention.
FIG. 3 is a schematic of 3 positive clinical specimens.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The most key concept of the invention is as follows: a bacterial drug-resistant gene quantum dot chip nucleic acid detection kit with high flux, high sensitivity and high specificity is established by utilizing the optical characteristics and the gene chip characteristics of a quantum dot material, and can simultaneously detect 13 common gram-negative and gram-positive bacterial drug-resistant genes.
The detection spectrum of the nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip is as follows: mecA, VanA, VanB, KPC-2, CTX-M-1, CTX-M-9, VIM-1, IMP-1/4, OXA-23, OXA-51, NDM-1, DHA-1 and MCR-1.
The invention designs detection primers and probes according to nucleic acid information of different drug-resistant genes, screens the primers and probes according to sensitivity, performs combination and related optimization of singleplex to multiplex of the primers meeting the requirements, determines the optimal primer combination and different reaction systems, and reduces the related influence among the primers. Finally, determining the primer combination form of each reaction system, and dividing the reaction system into 2 tubes (6-9 times of PCR).
Example 1:
preparation and application of quantum dot nucleic acid detection kit for urinary tract infection pathogens
Firstly, a quantum dot nucleic acid detection principle:
and performing molecular hybridization on the nucleic acid amplification product with the biotin label and a probe on a detection membrane strip, combining the biotin and a quantum dot coupled with streptavidin, and observing whether each site has a light signal by the detection membrane strip through a fluorescence detector to judge whether the probe is hybridized with the nucleic acid product, thereby determining whether the sample contains related target nucleic acid.
The capture probe is characterized in that amino groups are marked at the 3 'end or the 5' end of oligonucleotide single-stranded DNA, a spacing arm is arranged between the amino groups and the oligonucleotide single-stranded DNA, the spacing arm is a fatty acid C (n) chain or an oligo dT (n) chain or a combination of the fatty acid C (n) chain and the oligo dT (n), the number of the fatty acid C (n) chain n is 1-12, and the number of the oligo dT (n) n is 1-30.
The detection membrane strip is made of a nylon membrane, and capture probes (1-50uM) with certain concentration are dotted on the activated nylon membrane and distributed on the nylon membrane in a microarray mode.
The quantum dots are quantum dots (CdSe/ZnS) with a plurality of coupled streptavidin on the surface, and the number of the specifically coupled streptavidin is more than or equal to 1. The excitation wavelength of the quantum dot is 200-500nm, and the emission wavelength of the quantum dot is 400-700 nm. The size of the quantum dots is 1-200 nm.
The 5 'end of the nucleic acid amplification product is provided with a biotin label, specifically, the 5' end of one primer of the nucleic acid amplification is modified with the biotin label, the primer is connected with biotin to form a spacer arm, the spacer arm is a fatty acid C (n) chain or an oligo dT (n) chain or a combination of the fatty acid C (n) chain and the oligo dT (n), the number of the fatty acid C (n) chain n is 1-12, and the number of the oligo dT (n) n is 1-30.
The nucleic acid amplification method comprises polymerase chain reaction (such as PCR) and isothermal amplification (such as TMA/RPA/LAMP).
Quantum dot nucleic acid detection process:
1) firstly, carrying out nucleic acid amplification by using a plurality of pairs of primers, wherein the 5' end of one primer in one pair of primers for certain gene amplification is modified with biotin, the primer is connected with biotin to form a spacer arm, the spacer arm is a fatty acid C (n) chain or oligodT (n) chain or a combination of the fatty acid C (n) chain and oligodT (n), the number of the fatty acid C (n) chain n is 1-12, and the number of the oligodT (n) n is 1-30.
2) After the nucleic acid amplification, the product is subjected to a nucleic acid denaturation treatment by high-temperature heat denaturation. The high-temperature heating denaturation is more than 95 ℃.
3) And adding the denatured product and the detection membrane strip into a hybridization solution preheated to a certain temperature (40-55 ℃) in advance for hybridization, wherein the hybridization time is 30min-2 h. The hybridization solution was 2 × SSC with 0.1% SDS.
4) After hybridization, transferring the detection membrane strip into a washing solution preheated to a certain temperature (40-55 ℃) in advance for washing for 5-15 min. The wash was 0.5 SSC with 0.1% SDS.
5) After washing, removing the washing solution, adding the washing solution into an incubation solution with a certain temperature for incubation, wherein the incubation time is 5-30min, the temperature is 20-37 ℃, and the incubation solution is formed by adding 0.01nM-5nMSA-QD quantum dots (the excitation wavelength is 200-500nM and the emission wavelength is 400-700nM) into 2 x SSC and 0.1% SDS. The size of the quantum dots is 1-200 nm.
6) After the incubation is finished, removing the incubation liquid, and adding a certain amount of washing liquid for washing for 5-15 min. The wash was 0.5 SSC with 0.1% SDS.
7) And after washing, placing the detection membrane strip in a fluorescence instrument for fluorescence detection.
Second, design and screening of primers
The method comprises the steps of inquiring and downloading sequences of various drug resistance detection genes mecA, VanA, VanB, KPC-2, CTX-M-1, CTX-M-9, VIM-1, IMP-1/4, OXA-23, OXA-51, NDM-1, DHA-1 and MCR-1 in a NCBI database of a bioinformatics website, finding out regions with the highest target homology through BLAST comparison, and designing amplification primers. Primers with sensitivity meeting the requirements are screened by a large number of experimental tests (single amplification and multiple combined amplification). The specific detection primer sequences and sequence numbers are as follows:
primer VANAF: TGGCAAGTCAGGTGAAGATG (SEQ No. 1);
primer VANAR: CTAGACCTCTACAGCCGAGC (SEQ No. 2);
primer VANBF: CGTATTGACGTGGCTTTCCCG (SEQ No. 3);
primer VANBR: CGTGGATAGCGGCTGTACGAT (SEQ No. 4);
primer MEF GAAATGACTGAACGTCCG (SEQ No. 5);
primer MER: CATAACCTAATAGATGTGAAGTC (SEQ No. 6);
primer NDF: AGCCGCTGCATTGATGCTGAG (SEQ No. 7);
primer NDR: CGCGGTTGCTGGTTCGACC (SEQ No. 8);
primer CT9F: GCGTTGCAGTACAGCGACAATA (SEQ No. 9);
primer CT9R: ATATCATTGGTGGTGCCGTA (SEQ No. 10);
primer CT1F: GGACGATGTCACTGGCTGAGC (SEQ No. 11);
primer CT1R: CACAACCCAGGAAGCAGGCAGTC (SEQ No. 12);
primer MCF: GGCCTGCGTATTTTAAGCG (SEQ No. 13);
primer MCR: ATAGACACCGTTCTCACCCAG (SEQ No. 14);
primer VIMF: CGAGTGGTGAGTATCCGACAG (SEQ No. 15);
primer VIMR: GTCGTCATGAAAGTGCGTG (SEQ No. 16);
primer O5F: GGCAACCACCACAGAAGTATT (SEQ No. 19);
primer O5R: CCAGCCTACTTGTGGGTCTAC (SEQ No. 20);
primer O2F: ATCTATATGGTAATGCTCTAAGC (SEQ No. 21);
primer O2R: ACCTCTTGAATAGGCGTAACCT (SEQ No. 22);
CAAAACAGCCGGTCACTG (SEQ No. 23);
primer AMR: AGCAACTGCTCATACGGCAT (SEQ No. 24);
primer KPF: AACTGACACTGGGCTCTGCACT (SEQ No. 25);
primer KPR: AATCCCTCGAGCGCGAGTCTA (SEQ No. 26);
GTAGCATTGCTACCGCAGCAG (SEQ No.27) as primer;
CACTACGTTATCTGGAGTGTGT (SEQ No. 28);
each reverse primer is modified with a biotin tag. Wherein, the internal control IC amplification primer HBBF1: TATGGTTGGGATAAGGCTGG (SEQ No.17) is screened out; primer HBBR1: CGAGCTTAGTGATACTTGTG (SEQ No. 18);
third, confirmation of amplification reaction liquid System
Determining the composition of each reaction liquid system through a large number of multiple combination tests and system optimization tests, wherein the specific conditions are as follows:
the reaction system (48 parts by weight) of reaction solution I is shown in Table 1;
TABLE 1
The reaction system (48 parts by weight) of reaction solution II is shown in Table 2;
TABLE 2
The detection of each sample needs to be carried out by amplifying three reaction systems at the same time, each reaction system has 21ul, the extracted nucleic acid template is 4ul, and the total volume is 25 ul.
Fourthly, determination of PCR reaction program
Through a large number of test tests, the amplification program can effectively amplify the primers in each reaction system to the maximum extent, and the detection sensitivity of each drug-resistant gene reaches 100copies/ul genome concentration. The specific procedure is as follows (using the touchdown PCR procedure) as in table 4;
TABLE 4
Design of capture probe
The sequences of the drug-resistant genes mecA, VanA, VanB, KPC-2, CTX-M-1, CTX-M-9, VIM-1, IMP-1/4, OXA-23, OXA-51, NDM-1, DHA-1 and MCR-1 are inquired and downloaded in a NCBI database of a bioinformatics website. The BLAST comparison finds out the area with the highest target specificity, and simultaneously considers that each capture probe can carry out hybridization test design probes at the same hybridization temperature, and through a large number of sensitivity test tests and specificity tests, the probe sequences of each drug-resistant gene are determined, and the specific sequences and the serial numbers are as follows:
probe VANAP: TCAATAGCGCGGACGAATT (SEQ No. 29);
probe VANBP: GTGACAAACCGGAGGCGAGGAC (SEQ No. 30);
GGCATCGTTCCAAAGAATGTA (SEQ ID NO: 31);
GCTGGGTCGAACCAGCAA (SEQ ID NO: 32);
probe CT9P: GTTGGTGACGTGGCTCAAA (SEQ No. 33);
probe CT1P: AGCCGACGTTAAACACCGCCA (SEQ No. 34);
TGAAGGTAATGAGCTTGCCAA (SEQ No.35) as a probe MCP;
ACACAGCGGCACTTCTCGC (SEQ ID NO: 36) as a probe VIMP;
probe O5P: AAACGCTTCCATTTAGCCC (SEQ No. 37);
probe O2P: CTCTAAGCCGCGCAAATAC (SEQ No. 38);
probe AMP CAGCAGTATCGGCCTGTTTGGTG (SEQ ID NO: 39);
probe KPP GTTGATTGGCTAAAGGGAAA (SEQ No. 40);
probe IMPP: CATTTAGCGGAGTTAACTAT (SEQ No. 41);
probe ICP1: TTTGCTAATCATGTTCATACC (SEQ No. 42);
wherein the ICP1 probe is an internal control probe and is used for monitoring false negative in the process of extracting and amplifying the sample nucleic acid.
The 5' end of each probe was labeled with an amino group, and an oligo dT10 was located between the amino group and the oligonucleotide strand.
Sixth, preparation of detection membrane strip
Each capture probe is synthesized by a primer synthesis unit, then diluted by diluent to the required concentration, and then fixed on a nylon membrane through the condensation reaction of amino and carboxyl to prepare the detection membrane strip.
The layout of the test strips is shown in Table 5 below;
TABLE 5
The corresponding drug resistant genes on the membrane strip are shown in the following table 6:
TABLE 6
Seventh, determination of hybridization conditions
After PCR amplification is finished, 2-tube amplification products are mixed and subjected to denaturation treatment at 95 ℃ for 10min, and then hybridization, washing, incubation, washing and fluorescence detection are carried out. In the hybridization step, the hybridization temperature has a great influence on the interpretation of the result, the hybridization temperature is too low, non-specific capture can occur to cause false positive, the hybridization temperature is too high, the binding rate of the target product and the capture probe can be reduced, and finally the sensitivity is reduced to cause false negative. The subsequent washing temperature, the length of incubation time, and the concentration of SA-QD in the incubation solution will also have the same effect on the results.
And (3) hybridization:
and adding the denatured PCR product and the detection membrane strip into 1ml of hybridization solution which is pre-incubated to 48 ℃, and carrying out hybridization for 1.5h by gentle shaking at 48 ℃. While preheating 1ml of the wash liquor to 48 ℃.
The hybridization solution was 2 × SSC with 0.1% SDS. The wash was 0.5 SSC with 0.1% SDS.
Washing:
and taking out the detection membrane strip, transferring the detection membrane strip into a washing solution preheated to 48 ℃, and washing for 5min by shaking.
And (3) incubation:
1uM SA-QD was added to 1ml of the hybridization solution to prepare an incubation solution. Transferring the detection membrane strip into an incubation solution, incubating at room temperature, and shaking gently for 30 min.
Washing:
and taking out the detection membrane strip, transferring the detection membrane strip into a washing solution, and washing the detection membrane strip for 5min by gentle shaking at room temperature.
In summary, the present embodiment provides a nucleic acid detection kit and a detection method for a bacterial drug-resistant gene quantum dot chip, where the kit includes the detection membrane strip, a fluorescence detection solution and a reaction solution, and the detection membrane strip includes a nylon membrane and a capture probe fixed on the nylon membrane; the fluorescence detection solution comprises quantum dots which are used for marking the surface of the capture probe and are coupled with streptavidin; the reaction solution comprises: reaction liquid I and reaction liquid II.
The use process of the nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip in the embodiment is as follows:
1. and mixing the reaction solution I, the reaction solution II and the reaction solution III according to a ratio of 20.75 ul: mixing at a ratio of 0.25 ul.
2. 4ul of sample nucleic acid was added to the mixed reaction solution, and the mixture was placed in a PCR apparatus for PCR amplification. The amplification procedure is as in table 4;
3. after PCR amplification, the products are mixed and then denatured, and the nucleic acid denaturation method includes high-temperature heating denaturation. The high-temperature heating denaturation is more than 95 ℃.
4. And adding the denatured product and the detection membrane strip into a hybridization solution preheated to 48 ℃ in advance for hybridization, wherein the hybridization time is 30min-2 h. The hybridization solution was 2 × SSC with 0.1% SDS.
5. And after hybridization, transferring the detection membrane strip into a washing solution preheated to 48 ℃ in advance for washing for 5-15 min. The wash was 0.5 SSC with 0.1% SDS.
6. After washing, removing the washing solution, adding the washing solution into an incubation solution at room temperature for incubation for 5-30min, wherein the incubation solution is formed by adding SA-QD quantum dots (the excitation wavelength is 200-500nM, and the emission wavelength is 400-700nM) at the concentration of 0.01-5 nM into 2-SSC and 0.1% SDS. The size of the quantum dots is 1-200 nm.
7. After the incubation is finished, removing the incubation liquid, and adding a certain amount of washing liquid for washing for 5-15 min. The wash was 0.5 SSC with 0.1% SDS.
8. And after washing, placing the detection membrane strip in a fluorescence instrument for fluorescence detection.
Example 2
The effect verification analysis of the kit for detecting the quantum dot nucleic acid of the urinary tract infection pathogen
1. Sensitivity detection
The recombinant plasmids of the drug-resistant genes with the calibrated concentration are sequentially subjected to gradient dilution (1000 copies/. mu.L and 100 copies/. mu.L), and DNAs with the concentrations are respectively detected by a PCR-quantum dot fluorescence detection method, so that a positive result is judged if obvious bright spots appear, and a negative result is judged if no spots exist. The assay was tested according to the kit protocol in example 1. The detection results are shown in fig. 1 and fig. 2.
2. Clinical sample testing
Clinical blood culture positive result blood culture clinical sample detection is selected, genomic DNA extraction is firstly carried out, 1.2M sorbitol and circulating free nucleic acid extraction kit (magnetic bead method) produced by Hangzhou Chiji Biotechnology limited company and muramidase produced by Tiangen biochemistry are adopted as specific extraction reagents, and the extraction process is as follows:
1) taking 200ul blood culture solution to 1.5ml EP tube, centrifuging at 12000r for 5min, and removing supernatant;
2) adding 600ul 1.2M sorbitol buffer, re-suspending and precipitating, adding 5ul muramidase, and water-bathing at 30 deg.C for 30 min;
3) after water bath, centrifuging at 12000r for 5min, removing supernatant, and adding 200ul of normal saline for heavy suspension precipitation;
4) adding 300ul of lysis binding solution into an EP tube, adding 5ul of mag, mixing uniformly, and incubating at normal temperature for 10 min;
5) placing the EP tube on a magnetic separator, separating and discarding supernatant;
6) adding 1ml of washing solution into an EP tube, and fully washing;
7) placing the EP tube on a magnetic separator, separating and discarding supernatant, and air drying for 2-5 min;
8) adding 60ul of eluent into an EP tube, resuspending the magnetic beads, and heating at 56 deg.C for 5 min;
9) the EP tube was placed on a magnetic separator, the supernatant was separated and transferred to a clean EP tube and stored at-20 ℃ until use.
A reaction system was prepared in accordance with example 1, and then reaction solutions were dispensed in an amount of 21 ul/reaction, 4ul of the extracted nucleic acid was added to each reaction solution, PCR amplification was performed in accordance with the method of example 1, and hybridization test was performed on the amplified product in accordance with the method of example 1.
The results of the test are shown in FIG. 3. Clinical samples were positive for KPC-2 and OXA-23, and positive for VanB; one example is NDM-1 positive.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Hangzhou Qianji Biotechnology Co., Ltd
Hangzhou Boxin Biotechnology Co., Ltd
<120> bacterial drug-resistant gene quantum dot chip nucleic acid detection kit and detection method
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Claims (10)
1. A nucleic acid detection kit for a bacterial drug-resistant gene quantum dot chip is characterized by comprising a detection membrane strip, fluorescent detection liquid and reaction liquid, wherein the detection membrane strip comprises a nylon membrane and a capture probe fixed on the nylon membrane; the fluorescence detection solution comprises quantum dots which are used for marking the surface of the capture probe and are coupled with streptavidin; the reaction solution comprises: reaction liquid I, reaction liquid II and reaction liquid III;
the reaction solution I comprises the following detection primers:
TGGCAAGTCAGGTGAAGATG primer VANAF, SEQ No. 1;
CTAGACCTCTACAGCCGAGC, SEQ ID No. 2;
CGTATTGACGTGGCTTTCCCG, SEQ ID No. 3;
CGTGGATAGCGGCTGTACGAT, SEQ ID No. 4;
GAAATGACTGAACGTCCG for primer MEF, SEQ No. 5;
CATAACCTAATAGATGTGAAGTC primer MER, SEQ No. 6;
AGCCGCTGCATTGATGCTGAG primer NDF, SEQ ID No. 7;
CGCGGTTGCTGGTTCGACC primer NDR, SEQ No. 8;
primer CT9F: GCGTTGCAGTACAGCGACAATA, SEQ No. 9;
primer CT9R: ATATCATTGGTGGTGCCGTA, SEQ No. 10;
primer CT1F: GGACGATGTCACTGGCTGAGC, SEQ No. 11;
primer CT1R: CACAACCCAGGAAGCAGGCAGTC, SEQ No. 12;
GGCCTGCGTATTTTAAGCG for primer MCF, SEQ No. 13;
ATAGACACCGTTCTCACCCAG primer MCR, SEQ No. 14;
CGAGTGGTGAGTATCCGACAG primer VIMF, SEQ No. 15;
GTCGTCATGAAAGTGCGTG, SEQ ID No. 16;
primer HBBF1: TATGGTTGGGATAAGGCTGG, SEQ No. 17;
primer HBBR1: CGAGCTTAGTGATACTTGTG, SEQ No. 18;
the reaction solution II comprises the following detection primers:
primer O5F: GGCAACCACCACAGAAGTATT, SEQ No. 19;
primer O5R: CCAGCCTACTTGTGGGTCTAC, SEQ No. 20;
primer O2F: ATCTATATGGTAATGCTCTAAGC, SEQ No. 21;
primer O2R: ACCTCTTGAATAGGCGTAACCT, SEQ No. 22;
CAAAACAGCCGGTCACTG primer AMF, SEQ No. 23;
primer AMR AGCAACTGCTCATACGGCAT, SEQ No. 24;
AACTGACACTGGGCTCTGCACT primer KPF, SEQ No. 25;
AATCCCTCGAGCGCGAGTCTA primer KPR, SEQ No. 26;
GTAGCATTGCTACCGCAGCAG primer, SEQ No. 27;
CACTACGTTATCTGGAGTGTGT primer, SEQ No. 28;
primer HBBF1: TATGGTTGGGATAAGGCTGG, SEQ No. 17;
primer HBBR1: CGAGCTTAGTGATACTTGTG, SEQ No. 18;
the reaction solution III is Hotstart Taq DNA polymerase.
2. The nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip according to claim 1, wherein the reaction solution I comprises the following components:
water: 9.7 mu L of the suspension liquid,
10×PCR buffer：2.5μL，
20mM dN(U)TP：0.25μL，
mu.M of primers having nucleotide sequences shown in SEQ ID Nos. 1 to 16, each 0.5. mu.L,
mu.L of each primer having a 10. mu.M nucleotide sequence shown in SEQ ID Nos. 17-18,
1U/. mu.L UNG enzyme: 0.1 mu L;
the reaction solution II comprises the following components in percentage by weight:
water: 12.7 mu L of the mixture is prepared,
10×PCR buffer：2.5μL，
20mM dN(U)TP：0.25μL，
mu.M of each primer having a nucleotide sequence shown in SEQ ID Nos. 19 to 28 is 0.5. mu.L,
mu.L of each primer having a 10. mu.M nucleotide sequence shown in SEQ ID Nos. 17-18,
1U/. mu.L UNG enzyme: 0.1. mu.L.
3. The kit for detecting the nucleic acid of the bacterial drug-resistant gene quantum dot chip according to claim 1, wherein the capture probe comprises:
probe VANAP: TCAATAGCGCGGACGAATT, SEQ ID No. 29;
GTGACAAACCGGAGGCGAGGAC for probe VANBP, SEQ No. 30;
GGCATCGTTCCAAAGAATGTA for probe MEP, SEQ No. 31;
GCTGGGTCGAACCAGCAA, SEQ ID No. 32;
probe CT9P: GTTGGTGACGTGGCTCAAA, SEQ ID No. 33;
probe CT1P: AGCCGACGTTAAACACCGCCA, SEQ ID No. 34;
TGAAGGTAATGAGCTTGCCAA, namely SEQ No. 35;
ACACAGCGGCACTTCTCGC, SEQ ID No. 36;
probe O5P: AAACGCTTCCATTTAGCCC, SEQ ID No. 37;
probe O2P: CTCTAAGCCGCGCAAATAC, SEQ ID No. 38;
CAGCAGTATCGGCCTGTTTGGTG for probe AMP, SEQ ID No. 39;
GTTGATTGGCTAAAGGGAAA, SEQ No. 40;
the probe IMPP: CATTTAGCGGAGTTAACTAT, SEQ No. 41.
4. The nucleic acid detection kit for the bacterial drug-resistant gene quantum dot chip according to claim 1, wherein the detection membrane strip further comprises an internal control probe for monitoring the extraction and amplification of the sample nucleic acid: ICP1: TTTGCTAATCATGTTCATACC, SEQ No. 42.
5. The kit for detecting the nucleic acid of the bacterial drug-resistant gene quantum dot chip according to claim 1, wherein the capture probe is oligonucleotide single-stranded DNA, the 3 'end or the 5' end of the oligonucleotide single-stranded DNA is labeled with an amino group, an intermediate arm is connected between the oligonucleotide single-stranded DNA and the amino group, the intermediate arm is one or a combination of two of a fatty acid carbon chain and oligo dT (n), the length of the fatty acid carbon chain is 1-12 carbon atoms, and n in the oligo dT (n) is an integer from 1-30.
6. The kit for detecting the nucleic acid of the bacterial drug-resistant gene quantum dot chip according to claim 1, wherein a biotin label is modified at the 5' end of a reverse primer in the detection primer, an intermediate arm is connected between the detection primer and the biotin, the intermediate arm is one or a combination of two of a fatty acid carbon chain and oligo dT (n), the length of the fatty acid carbon chain is 1-12 carbon atoms, and n in the oligo dT (n) is an integer of 1-30.
7. The nucleic acid detection kit for a bacterial drug-resistant gene quantum dot chip as claimed in claim 1, wherein the excitation wavelength of the quantum dot is 200-500nm, the emission wavelength of the quantum dot is 400-700nm, and the size of the quantum dot is 1-200 nm.
8. The kit for detecting the nucleic acid of the bacterial drug-resistant gene quantum dot chip according to claim 1, wherein the quantum dot is a CdSe/ZnS core-shell quantum dot.
9. A bacterial drug-resistant gene quantum dot chip nucleic acid detection method, which is characterized in that the bacterial drug-resistant gene quantum dot chip nucleic acid detection kit of any one of claims 1 to 8 is adopted for detection.
10. The method for detecting the nucleic acid of the bacterial drug-resistant gene quantum dot chip according to claim 9, wherein the conditions for PCR amplification of the PCR reaction solution are set as follows:
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876508A (en) * | 2020-09-01 | 2020-11-03 | 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) | Fluorescent quantitative PCR kit for detecting Escherichia coli CTX-M gene |
| CN115725570A (en) * | 2022-11-02 | 2023-03-03 | 北京金匙医学检验实验室有限公司 | Nanopore sequencing-based method for detecting drug-resistant/virulence genes of blood sample |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952875A (en) * | 2011-08-31 | 2013-03-06 | 宁波市第二医院 | Bacterium drug-resistant gene detection method, gene chip and kit |
| CN106884037A (en) * | 2015-12-16 | 2017-06-23 | 博奥生物集团有限公司 | A kind of gene chip kit of detection bacterium drug resistant gene |
| CN107130016A (en) * | 2016-12-28 | 2017-09-05 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | VRE/MRSA/KPC/NDM 1 detects the preparation and use of genetic chip |
| CN108660189A (en) * | 2018-05-30 | 2018-10-16 | 杭州千基生物科技有限公司 | A kind of kit of quantum dot detection of nucleic acids for urinary tract infections pathogen |
| CN109196122A (en) * | 2016-03-07 | 2019-01-11 | 奥普根股份有限公司 | For determining the method and system of antibiotics sensitivity |
-
2019
- 2019-10-28 CN CN201911030693.8A patent/CN110904249B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952875A (en) * | 2011-08-31 | 2013-03-06 | 宁波市第二医院 | Bacterium drug-resistant gene detection method, gene chip and kit |
| CN106884037A (en) * | 2015-12-16 | 2017-06-23 | 博奥生物集团有限公司 | A kind of gene chip kit of detection bacterium drug resistant gene |
| CN109196122A (en) * | 2016-03-07 | 2019-01-11 | 奥普根股份有限公司 | For determining the method and system of antibiotics sensitivity |
| CN107130016A (en) * | 2016-12-28 | 2017-09-05 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | VRE/MRSA/KPC/NDM 1 detects the preparation and use of genetic chip |
| CN108660189A (en) * | 2018-05-30 | 2018-10-16 | 杭州千基生物科技有限公司 | A kind of kit of quantum dot detection of nucleic acids for urinary tract infections pathogen |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876508A (en) * | 2020-09-01 | 2020-11-03 | 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) | Fluorescent quantitative PCR kit for detecting Escherichia coli CTX-M gene |
| CN115725570A (en) * | 2022-11-02 | 2023-03-03 | 北京金匙医学检验实验室有限公司 | Nanopore sequencing-based method for detecting drug-resistant/virulence genes of blood sample |
| CN115725570B (en) * | 2022-11-02 | 2023-10-03 | 北京金匙医学检验实验室有限公司 | Nanopore sequencing-based blood sample drug resistance/virulence gene detection method |
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