CN106884037B - Gene chip kit for detecting bacterial drug resistance gene - Google Patents

Gene chip kit for detecting bacterial drug resistance gene Download PDF

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CN106884037B
CN106884037B CN201510941149.4A CN201510941149A CN106884037B CN 106884037 B CN106884037 B CN 106884037B CN 201510941149 A CN201510941149 A CN 201510941149A CN 106884037 B CN106884037 B CN 106884037B
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张岩
王辉
姜可伟
王杉
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Abstract

The invention discloses a gene chip kit for detecting a drug-resistant gene of bacteria. The kit provided by the invention contains a primer pair group for detecting the drug resistance gene of bacteria, and the primer pair group consists of 13 primer pairs, and the sequence of the primer pair is the sequence 1-26 in a sequence table; meanwhile, the kit also comprises a hybridization chip on which 13 single-stranded DNA probes shown in sequences 27-39 are fixed. The kit provided by the invention supports high-throughput, rapid and accurate detection of a plurality of bacterial drug resistance genes, screens Chinese population, and can effectively play roles in accurate diagnosis, drug resistance traceability, drug resistance control and the like, thereby reducing the usage amount of antibiotics and reducing the generation of drug resistance.

Description

Gene chip kit for detecting bacterial drug resistance gene
Technical Field
The invention belongs to the technical field of nucleic acid amplification, and relates to a gene chip kit for detecting a drug-resistant gene of bacteria.
Background
The detection of the drug-resistant gene of the bacteria has important clinical significance. The clinical diagnosis has the defects of large dosage, high starting point and random types of antibiotics, thereby causing the generation and the prevalence of drug-resistant strains. At present, the detection method for drug-resistant genes is still more traditional, and generally comprises a method for amplifying corresponding genes by Polymerase Chain Reaction (PCR) after physiological and biochemical tests, serological tests, drug sensitivity tests and the like, but the detection method has the disadvantages of long time, complex operation, slow report result and low flux, and the strains are often identified first to correspondingly carry out subsequent tests such as drug sensitivity and the like, so the detection method is difficult to adapt to the requirements of clinical treatment. Therefore, the rapid high-flux molecular diagnosis technology detection is developed, the rapid diagnosis can be assisted, and the timely and accurate medication can be guided.
The biochip technology is a high and new technology which is rapidly developed and matured in the life science field in recent years, and a micro biochemical analysis system which is constructed on the surface of a solid-phase chip mainly through a micro-processing technology and a microelectronic technology can realize accurate, rapid and large-amount information detection on cells, proteins, nucleic acids and other various biological components. The main characteristics of biochips are high throughput, miniaturization and automation.
Disclosure of Invention
The first purpose of the invention is to provide a primer pair group for detecting bacterial drug resistance genes.
The primer pair group for detecting the bacterial drug resistance gene provided by the invention consists of the following 13 primer pairs:
1) the primer set shown in (a1) or (a2) below was designated as primer set 1:
(a1) a primer pair consisting of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table;
(a2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and the sequence 2 in the sequence table and has the same function as the primer pair in (a 1);
2) the primer set shown in (b1) or (b2) below was designated as primer set 2:
(b1) a primer pair consisting of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table;
(b2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 3 and the sequence 4 in the sequence table and has the same function as the primer pair in the (b 1);
3) the primer set shown in (c1) or (c2) below was designated as primer set 3:
(c1) a primer pair consisting of two single-stranded DNA molecules shown in a sequence 5 and a sequence 6 in a sequence table;
(c2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and the sequence 6 in the sequence table and has the same function as the primer pair in the step (c 1);
4) the primer set shown in (d1) or (d2) below was designated as primer set 4:
(d1) a primer pair consisting of two single-stranded DNA molecules shown in a sequence 7 and a sequence 8 in a sequence table;
(d2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequences 7 and 8 in the sequence table and has the same function as the primer pair in the step (d 1);
5) the primer set shown below (e1) or (e2) was designated as primer set 5:
(e1) a primer pair consisting of two single-stranded DNA molecules shown in a sequence 9 and a sequence 10 in a sequence table;
(e2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 9 and the sequence 10 in the sequence table and has the same function as the primer pair in the (e 1);
6) the primer set shown in (f1) or (f2) below was designated as primer set 6:
(f1) a primer pair consisting of two single-stranded DNA molecules shown in a sequence 11 and a sequence 12 in a sequence table;
(f2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 11 and the sequence 12 in the sequence table and has the same function as the primer pair in the step (f 1);
7) the primer set shown below (g1) or (g2) was designated as primer set 7:
(g1) a primer pair consisting of two single-stranded DNA molecules shown as a sequence 13 and a sequence 14 in a sequence table;
(g2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence table of the sequence 13 and the sequence 14 and has the same function as the primer pair in the step (g 1);
8) the primer set shown below (h1) or (h2) was designated as primer set 8:
(h1) a primer pair consisting of two single-stranded DNA molecules shown as a sequence 15 and a sequence 16 in a sequence table;
(h2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence table 15 and the sequence table 16 and has the same function as the primer pair in the (h 1);
9) the primer pair shown in (i1) or (i2) below was designated as primer pair 9:
(i1) a primer pair consisting of two single-stranded DNA molecules shown in a sequence 17 and a sequence 18 in a sequence table;
(i2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence table of the sequence 17 and the sequence 18 and has the same function as the primer pair in the (i 1);
10) the primer set shown in (j1) or (j2) below was designated as primer set 10:
(j1) a primer pair consisting of two single-stranded DNA molecules shown as a sequence 19 and a sequence 20 in a sequence table;
(j2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence table of the sequence 19 and the sequence 20, and has the same function as the primer pair in the (j 1);
11) the primer set shown below (k1) or (k2) was designated as primer set 11:
(k1) a primer pair consisting of two single-stranded DNA molecules shown as a sequence 21 and a sequence 22 in a sequence table;
(k2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 21 and the sequence 22 in the sequence table and has the same function as the primer pair in the (k 1);
12) the primer set shown below (l1) or (l2) was designated as primer set 12:
(l1) a primer pair consisting of two single-stranded DNA molecules shown by a sequence 23 and a sequence 24 in the sequence table;
(l2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence table for the sequence 23 and the sequence 24, and has the same functions as the primer pair in (l 1);
13) the primer set shown in (m1) or (m2) below was designated as primer set 13:
(m1) a primer pair consisting of two single-stranded DNA molecules shown by a sequence 25 and a sequence 26 in the sequence table;
(m2) is composed of two single-stranded DNA molecules shown by sequences obtained by replacing and/or deleting and/or adding one or more nucleotides in the sequence table of the sequence 25 and the sequence 26, and the functions of the primer pairs are the same as those of the primer pair in (m 1).
Wherein, the primer pair 1 is used for amplifying the mecA gene; the primer pair 2 is used for amplifying a vanA gene; the primer pair 3 is used for amplifying the vanB gene; the primer pair 4 is used for amplifying blaDHA-1A gene; the primer pair 5 is used for amplifying blaOXA-23A gene; the primer pair 6 is used for amplifying blaOXA-24A gene; the primer pair 7 is used for amplifying blaOXA-58A gene; the primer pair 8 is used for amplifying blaKPC-1A gene; the primer pair 9 is used for amplifying blaIMP-4A gene; the primer pair 10 is used for amplifying blaVIM-8A gene; the primer pair 11 is used for amplifying blaNDM-1A gene; the primer pair 12 is used for amplifying blaCTX-M-1A gene; the primer pair13 for amplifying blaCTX-M-9A gene.
The second purpose of the invention is to provide a set of single-stranded DNA for detecting the drug resistance gene of bacteria.
The complete single-stranded DNA for detecting the bacterial drug resistance gene provided by the invention specifically comprises a probe set and a primer pair set; the probe set consists of 13 single-stranded DNA probes as follows: a single-stranded DNA probe 1 shown as a sequence 27 in a sequence table; a single-stranded DNA probe 2 shown as a sequence 28 in a sequence table; a single-stranded DNA probe 3 shown as a sequence 29 in a sequence table; a single-stranded DNA probe 4 shown as a sequence 30 in the sequence table; a single-stranded DNA probe 5 shown as a sequence 31 in a sequence table; a single-stranded DNA probe 6 shown as a sequence 32 in the sequence table; a single-stranded DNA probe 7 shown as a sequence 33 in the sequence table; a single-stranded DNA probe 8 shown in a sequence 34 in a sequence table; a single-stranded DNA probe 9 shown in a sequence 35 in a sequence table; a single-stranded DNA probe 10 shown as a sequence 36 in a sequence table; a single-stranded DNA probe 11 shown in a sequence 37 in a sequence table; a single-stranded DNA probe 12 shown in a sequence 38 in the sequence table; a single-stranded DNA probe 13 shown as a sequence 39 in the sequence table.
Wherein, the single-stranded DNA probe 1 is used for detecting the amplification result of the primer pair 1; the single-stranded DNA probe 2 is used for detecting the amplification result of the primer pair 2; the single-stranded DNA probe 3 is used for detecting the amplification result of the primer pair 3; the single-stranded DNA probe 4 is used for detecting the amplification result of the primer pair 4; the single-stranded DNA probe 5 is used for detecting the amplification result of the primer pair 5; the single-stranded DNA probe 6 is used for detecting the amplification result of the primer pair 6; the single-stranded DNA probe 7 is used for detecting the amplification result of the primer pair 7; the single-stranded DNA probe 8 is used for detecting the amplification result of the primer pair 8; the single-stranded DNA probe 9 is used for detecting the amplification result of the primer pair 9; the single-stranded DNA probe 10 is used for detecting the amplification result of the primer pair 10; the single-stranded DNA probe 11 is used for detecting the amplification result of the primer pair 11; the single-stranded DNA probe 12 is used for detecting the amplification result of the primer pair 12; the single-stranded DNA probe 13 is used for detecting the amplification result of the primer pair 13.
The third purpose of the invention is to provide a kit for detecting the drug resistance gene of bacteria.
The kit for detecting the bacterial drug resistance gene provided by the invention comprises the primer pair group and a hybridization chip; the hybridization chip is prepared according to a method comprising the following steps: the general formula is' NH2-(T)n13 single-stranded detection probes of the hybridization probe sequence "are immobilized on an aldehyde-modified solid support (e.g., a glass slide) by a reaction of an amino group and an aldehyde group, respectively, to obtain the hybridization chip; (T) in the formulanN is an integer of 5 to 30 inclusive; the 13 hybridization probe sequences corresponding to the 13 single-stranded detection probes in the general formula are respectively shown as sequences 27-39 in the sequence table.
Optionally, the kit can also contain a random primer which is marked by fluorescence; the sequence of the random primer is 5' -NX-3', N represents any of A, G, C and T, 6 ≦ X ≦ 15, and X is an integer (e.g., X ≦ 9), NXRepresents X consecutive deoxyribonucleotides.
The above-mentioned fluorescence-labeled random primers are used for fluorescence labeling of the amplification products of the primer pairs in the primer pair group. If necessary, the amplification product may be fluorescently labeled not with the fluorescently labeled random primer but with another method. For example, the 5' -end of one primer of each primer pair in the primer pair group is fluorescently labeled (e.g., TAMRA), and the fluorescently labeled primer is a primer present on the single-stranded DNA of the amplification product that can be hybridized by the corresponding probe.
The kit can also contain a hybridization solution; the hybridization solution contains fluorescence-labeled unrelated single-stranded DNA molecules; the unrelated single-stranded DNA molecule is a single-stranded DNA molecule which is not derived from the drug resistance gene of the bacteria.
The preparation method of the hybrid chip also comprises the step of fixing the surface chemical control probe QC, and/or the hybrid quality control probe PC, and/or the negative control probe BC on the aldehyde group modified solid phase carrier (such as a glass slide) through the reaction of amino and aldehyde groups;
the surface chemical quality control probe QC, the hybridization quality control probe PC and the negative control probe are single-stranded probes; one end of the surface chemical quality control probe QC is modified by amino, and the other end of the surface chemical quality control probe QC is provided with a fluorescent label (such as Hex); one end of the hybridization quality control probe PC is modified by amino and can be hybridized with the unrelated single-stranded DNA molecules in the hybridization solution; one end of the negative control probe BC is modified with an amino group and is not hybridizable to any single-stranded DNA molecule derived from the bacterial drug resistance gene.
Further, in the present invention, the structure composition of the surface chemistry control probe QC from 5 'end to 3' end is "NH2-TCACTTGCTTCCGTTGAGG-Hex "; the structural composition of the hybridization quality control probe PC from the 5 ' end to the 3 ' end is ' NH2-TTTTTTTTTTTTCCTCAACGGAAGCAAGTGAT "; the negative control probe BC has a structural composition from 5 ' end to 3 ' end as ' NH2-TTTTTTTTTTTTGTTGCTTCTGGAATGAGTTTGCT”。
The application of the primer pair group or the single-stranded DNA set or the kit in the following (a) or (b) also belongs to the protection scope of the invention:
(a) detecting or assisting in detecting bacterial drug resistance genes (for non-diagnostic purposes), or preparing products for detecting or assisting in detecting bacterial drug resistance genes;
(b) detecting or aiding detection of bacteria containing bacterial resistance genes (for non-diagnostic purposes), or preparing a product for detecting or aiding detection of bacteria containing bacterial resistance genes.
In the present invention, the bacterial drug resistance gene may specifically be at least one of: mecA gene, vanA gene, vanB gene, blaDHA-1Gene, blaOXA-23Gene, blaOXA-24Gene, blaOXA-58Gene, blaKPC-1Gene, blaIMP-4Gene, blaVIM-8Gene, blaNDM-1Gene, blaCTX-M-1Genes and blaCTX-M-9A gene.
The GenBank accession number of the mecA gene is AB221119.1 (update: 2006-5-19); the GenBank accession number of the vanA gene is M97297.1 (update: 2002-6-20); the GenBank accession number of the vanB gene is AY655711.1 (update: 2)005-11-7); the blaDHA-1GenBank accession number of the gene is EF406115.1 (update: 2007-8-17); the blaOXA-23GenBank accession number of the gene is JN665073.1 (update: 2011-11-7); the blaOXA-24GenBank accession number of the gene is JN207494.1 (update: 2011-11-27); the blaOXA-58GenBank accession number of the gene is EU107372.1 (update: 2007-9-11); the blaKPC-1GenBank accession number of the gene is AF297554.1 (update: 2001-3-26); the blaIMP-4GenBank accession number of the gene is AF244145.1 (update: 2001-2-27); the blaVIM-8GenBank accession number of the gene is AY524987.1 (update: 2004-11-5); the blaNDM-1GenBank accession number of the gene is JF503991.1 (update: 2012-12-11); the blaCTX-M-1GenBank accession number of the gene is AJ416342.1 (update: 2008-10-23); the blaCTX-M-9GenBank accession number of the gene is AJ416345.1 (update: 2005-4-15).
Correspondingly, the bacteria containing the mecA gene can be staphylococcus aureus specifically; the bacterium containing the vanA gene can be specifically enterococcus faecalis or enterococcus faecium; the bacteria containing the vanB gene can be specifically enterococcus faecalis or enterococcus faecium; containing said blaDHA-1The bacteria of the gene can be Klebsiella pneumoniae; containing said blaOXA-23The bacteria of the gene can be Acinetobacter baumannii; containing said blaOXA-24The bacteria of the gene can be Acinetobacter baumannii; containing said blaOXA-58The bacteria of the gene can be Acinetobacter baumannii; containing said blaKPC-1The bacteria of the gene can be Klebsiella pneumoniae or pseudomonas aeruginosa; containing said blaIMP-4The bacteria of the gene can be Acinetobacter baumannii; containing said blaVIM-8The bacteria of the gene can be Klebsiella pneumoniae or Acinetobacter baumannii; containing said blaNDM-1The bacterium of the gene may specifically be Escherichia coli; containing said blaCTX-M-1The genetic bacteria can be proteus mirabilis; containing said blaCTX-M-9The bacterium of the gene may specifically be Escherichia coli.
The application of the primer pair group or the complete set of single-stranded DNA in the preparation of the kit also belongs to the protection scope of the invention.
The invention researches the bacterial drug resistance gene with a great sample amount. The kit provided by the invention supports high flux, rapidly and accurately detects a plurality of drug-resistant genes, screens Chinese population, and can effectively play roles in accurate diagnosis, drug-resistant traceability, drug-resistant control and the like, thereby reducing the usage amount of antibiotics and the generation of drug resistance.
Drawings
FIG. 1 is a schematic diagram of the array layout of the hybridization chip (i.e., a schematic diagram of the chip probe layout).
FIG. 2 shows the result of the specificity analysis of the kit for detecting a bacterial drug resistance gene. The corresponding chip probe layout is shown in FIG. 1. Wherein, the DNA plasmid of the reference product corresponding to the detection result of the chip shown in A is ZL-CTX-M-1 (corresponding to bla)CTX-M-1Genes); the DNA plasmid of the reference product corresponding to the detection result of the chip shown in B is ZL-CTX-M-9 (corresponding to bla)CTX-M-9Genes); the DNA plasmid of the reference product corresponding to the detection result of the chip shown in C is ZL-DHA-1 (corresponding to bla)DHA-1Genes); d the DNA plasmid of the reference product corresponding to the detection result of the chip is ZL-IMP-4 (corresponding to bla)IMP-4Genes); e, the DNA plasmid of the reference product corresponding to the detection result of the chip is ZL-KPC-1 (corresponding to bla)KPC-1Genes); f, the DNA plasmid of the reference product corresponding to the detection result of the chip is ZL-NDM-1 (corresponding to bla)NDM-1Genes); g the detection result of the chip corresponds to a reference DNA plasmid ZL-OXA-23 (corresponding to bla)OXA-23Genes); h, the DNA plasmid of the reference corresponding to the detection result of the chip is ZL-OXA-24 (corresponding to bla)OXA-24Genes); the DNA plasmid of the reference product corresponding to the detection result of the chip shown in I is ZL-OXA-58 (corresponding to bla)OXA-58Genes); j, the reference product DNA plasmid corresponding to the detection result of the chip is ZL-vanA (corresponding to vanA gene); the reference product DNA plasmid corresponding to the chip detection result shown by K is ZL-vanB (corresponding to a vanB gene); the reference DNA plasmid corresponding to the detection result of the chip shown by L is ZL-VIM-8 (corresponding to bla)VIM-8Genes); the reference product DNA plasmid corresponding to the detection result of the chip shown by M is ZL-mecA (corresponding to the mecA gene).
FIG. 3 is a graph showing the results of chip detection of three clinical samples. Wherein, A is No. 1 clinical sample; b is No. 2 clinical sample; c is clinical sample No. 3. The corresponding chip probe layout is shown in FIG. 1.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of a kit for detecting a bacterial drug resistance Gene and use thereof
Assembly and preparation of kit for detecting bacterial drug resistance gene
1. Primers and hybridization probes designed for 13 bacterial drug resistance genes
Specific sequences of primers and single-stranded hybridization probes designed for the 13 bacterial resistance genes are shown in tables 1 and 2.
TABLE 1 primers designed for 13 bacterial drug resistance genes
Figure BDA0000880190920000071
TABLE 2 Single-stranded hybridization probes designed for 13 bacterial drug resistance genes
Figure BDA0000880190920000072
Figure BDA0000880190920000081
2. Immobilization of hybridization probes on hybridization chips
The hybridization chip is a substrate on which 13 kinds of single-stranded detection probes are immobilized, respectively. Each detection probe is an amino-modified oligonucleotide probe, and the general formula of the 13 single-stranded detection probes is' NH2TTTTTTTTTTTTTTT-hybridization probe sequences ", wherein the" hybridization probe sequences "are the 13 single-stranded sequences shown as sequences 27 to 39 in Table 2Hybridization probes ". Each of the detection probes was dissolved in a gene spotting solution (product of Boo Bio Inc., Cat. having Cat. No. CP.440010) to a final concentration of 10. mu.M, and spotting was repeated three times on an aldehyde-modified slide (product of Boo Bio Inc., Cat. having Cat. No. CP.420022), whereby 13 kinds of probes were immobilized on the hybridization chip by the reaction of amino groups with aldehyde groups. In addition, the surface chemical control probe QC, the hybrid control probe PC and the negative control probe BC were also immobilized on the hybrid chip by the reaction of the amino group with the aldehyde group. QC is a single-stranded oligonucleotide probe with a Hex label at one end and an amino group modification at the other end for observing the efficiency of chip spotting and immobilization, and the structure from the 5 'end to the 3' end of the single-stranded oligonucleotide probe is composed of NH2-TCACTTGCTTCCGTTGAGG-Hex. PC is a single-stranded oligonucleotide probe modified by amino group, can be hybridized with fluorescence-labeled unrelated single-stranded DNA molecule (C-PC) added in hybridization solution, is used for quality control in hybridization process, and has a structure from 5 'end to 3' end consisting of NH2-TTTTTTTTTTTTCCTCAACGGAAGCAAGTGAT. BC is an amino-modified oligonucleotide probe, can not hybridize with all sequences to be detected in a hybridization system, is used for observing whether non-specific hybridization exists or not, and has a structure from 5 'end to 3' end consisting of NH2-TTTTTTTTTTTTGTTGCTTCTGGAATGAGTTTGCT. FIG. 1 is a schematic diagram of the array layout of the hybrid chip.
3. Composition of kit for detecting bacterial drug resistance gene
The kit for detecting bacterial drug resistance genes (namely 13 bacterial drug resistance genes related in table 1 and table 2) provided by the invention comprises:
(1) 13 primer pairs shown in table 1 in step 1;
(2) 13 detection probes, a surface chemical quality control probe QC, a hybrid quality control probe PC and a negative control probe BC are fixed on the hybridization chip in the step 2;
(3) a fluorescence-labeled random primer; the sequence of the random primer is 5' -N9-3', N represents any of A, G, C and T, N9Represents 9 consecutive deoxyribonucleotides.
(4) Hybridizing liquid; the hybridization solution contains an unrelated single-stranded DNA molecule (C-PC) which is marked by fluorescence, and the nucleotide sequence of the unrelated single-stranded DNA molecule is 5'-ATCACTTGCTTCCGTTGAGG-3'.
Second, use method of kit for detecting bacterial drug resistance gene
1. Sample Polymerase Chain Reaction (PCR) amplification
Taking 1 microliter of DNA sample solution to be detected, taking the DNA sample solution as a template, and respectively adopting 13 primer pairs shown in the table 1 to carry out PCR amplification. The 20 μ L PCR amplification system was as follows: 10 XPCR buffer (containing Mg)2+)2 mu L of the solution; 1.6. mu.L of 2.5mM dNTP; 10 μ M of the forward primer 0.5 μ L; 10 μ M downstream primer 0.5 μ L; 1 mu L of template; 5U/. mu.L rTaq 0.2. mu.L; water was added to 20 μ L. The PCR amplification procedure was as follows: 5min at 94 ℃; (94 ℃ for 30 s; 55 ℃ for 30 s; 72 ℃ for 1min) for 30 cycles; 10min at 72 ℃. After the PCR reaction, 13 PCR amplification products were obtained in total.
2. Fluorescent labeling of PCR amplification products
Adding 5 mu L of each PCR amplification product into different sample tubes, adding 3 mu L of TAMRA fluorescence labeled 9N random primer (the nucleotide sequence is 5 '-N9-3', N represents any one of A, G, C and T, N9 represents 9 continuous deoxyribonucleotides, specifically a product of the company Limited in the engineering and biological engineering (Shanghai)) with the concentration of 100 mu M into each sample tube, replenishing water to 19 mu L, shaking and uniformly mixing, and performing instantaneous centrifugation; after denaturation at 95 ℃ on ice, 6. mu.L of a fluorescence labeling reaction system mix (composition: 10 XKlenow Buffer 2.5. mu.L; 5U/. mu.L Klenow enzyme 1. mu.L; 2.5mM dNTP 2.5. mu.L) was added. Fluorescence labeling was performed according to the following procedure: 90min at 37 ℃; 10min at 70 ℃. In total 13 parts of TAMRA fluorescently labeled product were obtained.
3. Purification of TAMRA fluorescently labeled product
The TAMRA fluorescently labeled product obtained in step 2 was purified using a nucleic Spin Gel and PCR Clean-up kit (product code: REF 740609. about.250) manufactured by Macherey-Nagel company, and the detailed procedures were performed according to the kit instructions.
4. Hybridization of
Melting hybridization buffer (product of Boaobio corporation, product catalog number is CP.440030) containing TAMRA fluorescence labeled unrelated single-stranded DNA molecule (5'-ATCACTTGCTTCCGTTGAGG-3') at 50 ℃, preparing 13 parts of hybridization mixed solution (wherein, the TAMRA fluorescence labeled product solution purified in the step 3 is 15 muL, and the hybridization buffer is 5 muL), adding the prepared 13 parts of hybridization mixed solution to the hybridization chip in the step one 2, each part of hybridization mixed solution corresponds to a complete hybridization chip, denaturing at 95 ℃ for 3min, immediately placing on ice, and hybridizing in a water bath at 50 ℃ for 2 h.
5. Cleaning of
Washing with two different washing solutions (washing solution I: 2 XSSC, 0.2% SDS, preheated to 50 deg.C; washing solution II: 0.2 XSSC, preheated to 50 deg.C) for 4min respectively in the order of washing solution I and washing solution II, placing the chip in a slide Washer-8 chip washer, selecting a centrifugation program, centrifuging (or centrifuging at 1000rpm for 2min), and spin-drying.
6. Scanning and result determination
Scanning is finished by using a Boo crystal core LuxScan 10K microarray chip scanner (a green channel is selected, the parameter range of 'Power' is set to be 50-90, and the parameter range of 'PMT' is set to be 500-900), and whether the DNA sample to be detected contains the 13 bacterial drug-resistant genes related in the table 1 and which one or more of the 13 bacterial drug-resistant genes are specifically contained is determined according to the scanning result by the following method: if a probe for detecting a certain bacterial drug-resistant gene detects a fluorescent signal at a fixed position on the chip, determining that the corresponding DNA sample to be detected contains the corresponding bacterial drug-resistant gene; otherwise, the bacterial strain does not contain the corresponding bacterial drug resistance gene. In addition, in the scanning process, software can endow different colors according to the detected fluorescent signal intensity, the color is from blue to white, the signal is gradually enhanced, and then the content of the drug-resistant gene of the target bacteria in the DNA sample to be detected can be preliminarily judged.
Example 2 specificity and sensitivity assay of kit for detecting bacterial drug resistance genes
Preparation of reference DNA plasmid
1. Construction of plasmid containing mecA Gene target fragment
The DNA fragment shown in position 1001-1926 of the mecA gene (GenBank: AB221119.1, update: 2006-5-19) was ligated to pGEM-T Easy Vector (product of promega corporation) to obtain recombinant plasmid ZL-mecA. And verified to be correct by sequencing.
2. Construction of plasmid containing vanA Gene target fragment
The DNA fragment at position 7000-7988 of the vanA gene (GenBank: M97297.1, update: 2002-6-20) was ligated to pGEM-T Easy Vector to obtain recombinant plasmid ZL-vanA. And verified to be correct by sequencing.
3. Construction of plasmid containing target fragment of vanB Gene
The DNA fragment shown at positions 14-1029 of the vanB gene (GenBank: AY655711.1, update: 2005-11-7) was ligated to pGEM-T Easy Vector to obtain recombinant plasmid ZL-vanB. And verified to be correct by sequencing.
4. Containing blaDHA-1Construction of plasmid for Gene target fragment
Will blaDHA-1The DNA fragment shown in 1 st-1113 th site of the gene (GenBank: EF406115.1, update: 2007-8-17) is connected to a pGEM-T Easy Vector to obtain a recombinant plasmid ZL-DHA-1. And verified to be correct by sequencing.
5. Containing blaOXA-23Construction of plasmid for Gene target fragment
Will blaOXA-23The DNA fragment shown at positions 97-899 of the gene (GenBank: JN665073.1, update: 2011-11-7) was ligated to pGEM-T Easy Vector to obtain recombinant plasmid ZL-OXA-23. And verified to be correct by sequencing.
6. Containing blaOXA-24Construction of plasmid for Gene target fragment
Will blaOXA-24The DNA fragment indicated at position 4168-4882 of the gene (GenBank: JN207494.1, update: 2011-11-27) was ligated to pGEM-T Easy Vector to obtain recombinant plasmid ZL-OXA-24. And verified to be correct by sequencing.
7. Containing blaOXA-58Construction of plasmid for Gene target fragment
Will blaOXA-58The DNA fragment shown in 42 th-835 th positions of the gene (GenBank: EU107372.1, update: 2007-9-11) is connected to a pGEM-T Easy Vector to obtain a recombinant plasmid ZL-OXA-58. And verified to be correct by sequencing.
8. Containing blaKPC-1Construction of plasmid for Gene target fragment
Will blaKPC-1The DNA fragment shown in 154-1003 of the gene (GenBank: AF297554.1, update: 2001-3-26) was ligated to pGEM-T Easy Vector to obtain recombinant plasmid ZL-KPC-1. And verified to be correct by sequencing.
9. Containing blaIMP-4Construction of plasmid for Gene target fragment
Will blaIMP-4The DNA fragment shown in positions 1-470 of the gene (GenBank: AF244145.1, update: 2001-2-27) was ligated to pGEM-T Easy Vector to obtain recombinant plasmid ZL-IMP-4. And verified to be correct by sequencing.
10. Containing blaVIM-8Construction of plasmid for Gene target fragment
Will blaVIM-8The DNA fragment at positions 89-798 of the gene (GenBank: AY524987.1, update: 2004-11-5) was ligated to pGEM-T Easy Vector to obtain recombinant plasmid ZL-VIM-8. And verified to be correct by sequencing.
11. Containing blaNDM-1Construction of plasmid for Gene target fragment
Will blaNDM-1The DNA fragment indicated in 118437-119168 of the gene (GenBank: JF503991.1, update: 2012-12-11) was ligated to pGEM-T Easy Vector to obtain the recombinant plasmid ZL-NDM-1. And verified to be correct by sequencing.
12. Containing blaCTX-M-1Construction of plasmid for Gene target fragment
Will blaCTX-M-1The DNA fragment shown in position 567-1373 of the gene (GenBank: AJ416342.1, update: 2008-10-23) was ligated to pGEM-T Easy Vector to obtain recombinant plasmid ZL-CTX-M-1. And verified to be correct by sequencing.
13. Containing blaCTX-M-9Construction of plasmid for Gene target fragment
Will blaCTX-M-9The DNA fragment shown in 218-958 of the gene (GenBank: AJ416345.1, update: 2005-4-15) was ligated to pGEM-T Easy Vector to obtain recombinant plasmid ZL-CTX-M-9. And verified to be correct by sequencing.
Second, specificity analysis of kit for detecting bacterial drug resistance gene
Diluting the 13 reference product DNA plasmids constructed in the step one to a concentration of 104And copying/mu L, respectively taking the diluted 13 reference product DNA plasmids as DNA samples to be detected, then carrying out operation according to the step two in the example 1, and detecting whether the DNA samples to be detected contain the target bacterial drug resistance genes, wherein the specific judgment method refers to the step two 6 in the example 1.
As shown in FIG. 2, it can be seen that for each of the reference DNA plasmids, only the spot on which the corresponding detection probe was immobilized was able to detect a significant fluorescence signal, and no fluorescence signal was detected at any other spot. The result shows that the kit for detecting the bacterial drug resistance gene provided by the invention has stronger specificity.
Third, sensitivity analysis of kit for detecting bacterial drug resistance gene
Respectively carrying out gradient dilution on the 13 reference product DNA plasmids constructed in the step one to obtain the DNA plasmids with the concentration of 10 in sequence4Copy/. mu.L, 103Copy/. mu.L, 102Copy/. mu.L, 101Copies/. mu.L of dilutions. And (3) respectively taking 13 reference product DNA plasmids with different dilutions as DNA samples to be detected, then carrying out operation according to the step two in the example 1, and detecting whether the DNA samples to be detected contain target bacterial drug resistance genes, wherein the specific judgment method refers to the step two 6 in the example 1.
The results show that: for each reference DNA plasmid, the concentration was 104Copy/. mu.L, 103Copy/. mu.L, 102Obvious fluorescent signals can be detected at the point of the corresponding detection probe at the copying/mu L; only at a concentration of 101No fluorescent signal was detected at the copy/. mu.L of the reference DNA plasmid. The result shows that the kit for detecting the bacterial drug resistance gene provided by the invention has higher sensitivity.
Example 3 detection of actual clinical samples
Type of clinical sample
The clinical samples used in this example were from the viscous pus of wounds collected in the people hospital of Beijing university (the principle of this collector's own volunteers), in total, in triplicate.
Second, extraction of DNA sample from clinical specimen
1. Taking 1-3mL of the clinical sample obtained in the step I;
2. adding 4 times of 4% (4g/100mL) of NaOH, shaking, and standing at room temperature for 30min for liquefaction;
3. taking 0.5mL of liquefied pus and 0.5mL of 4% (4g/100mL) of NaOH at room temperature for 10 min;
4. centrifuging at 12000 rpm for 15 min;
5. discarding the supernatant, adding 1mL of sterile normal saline, mixing uniformly, and centrifuging at 12000 rpm for 5 min;
6. discarding the supernatant, and using the precipitate for DNA extraction;
7. adding 50 μ L of nucleic acid extract (product of Boo biological group, Inc., product catalog number CP.360090), shaking thoroughly, mixing well to make the precipitate completely suspended;
8. transferring the suspension into a nucleic acid extraction tube, screwing a tube cover, and placing the tube cover on a nucleic acid extraction instrument or a vortex oscillator to oscillate for 5min at the maximum rotating speed;
9. heating in metal bath at 95 deg.C for 5 min;
10. centrifuge at 5000rpm for 1min, transfer the supernatant to a 1.5mL centrifuge tube and store at-20 ℃ until use.
Third, detection of actual clinical samples
And (3) taking the three clinical sample DNAs obtained in the second step as DNA samples to be detected, then operating according to the second step in the example 1, and detecting whether the DNA samples to be detected contain target bacteria drug resistance genes, wherein the specific judgment method refers to the second step 6 in the example 1.
The results are shown in FIG. 3, and it can be seen that the hybridization signals of the three samples are clear and single. The position of the probe is compared, and the signal obtained by detecting the No. 1 clinical sample is the drug-resistant gene blaCTX-M-9And blaKPC-1And the signal detected in No. 2 clinical sample is drug-resistant gene blaOXA-23、blaOXA-58And blaVIM-8And the signal detected in No. 3 clinical sample is drug-resistant gene blaCTX-M-1And blaDHA-1。
For further determination toIn view of the accuracy of the detection results of the present invention, the inventors of the present invention performed agarose gel electrophoresis and sequencing verification on the PCR amplification products of three clinical samples, and the results confirmed that: no. 1 clinical sample only adopts that amplification products of primer pairs CTX-M9-F/CTX-M9-R and KPC-F/KPC-R have obvious electrophoretic bands, amplification products of other primer pairs have no obvious electrophoretic band, and further sequencing of amplification products of primer pairs CTX-M9-F/CTX-M9-R and KPC-F/KPC-R shows that the sequences of the amplification products are bla in sequenceCTX-M-9218 th-KPC-1154-1003 of gene (GenBank: AF297554.1, update: 2001-3-26); no. 2 clinical sample has obvious electrophoresis bands only by using the amplification products of the primer pairs of OXA23-F/OXA23-R, OXA58-F/OXA58-R and VIM-F/VIM-R, but no obvious electrophoresis band is detected by the amplification products of other primer pairs, and the sequencing of the amplification products of the primer pairs of OXA23-F/OXA23-R, OXA58-F/OXA58-R and VIM-F/VIM-R respectively finds that the sequences of the amplification products are bla a in the orderOXA-2397 th to 899 th part of gene (GenBank: JN665073.1, update: 2011-11-7), blaOXA-5842-835 th and bla of gene (GenBank: EU107372.1, update: 2007-9-11)VIM-889-798 th position of gene (GenBank: AY524987.1, update: 2004-11-5); no. 3 clinical sample only adopts that amplification products of primer pairs CTX-M1-F/CTX-M1-R and DHA-F/DHA-R have obvious electrophoretic bands, amplification products of other primer pairs do not detect obvious electrophoretic bands, and further sequencing of amplification products of primer pairs CTX-M1-F/CTX-M1-R and DHA-F/DHA-R shows that the sequences of the amplification products are bla in sequenceCTX-M-1The 567-th-1373-th site and bla of the gene (GenBank: AJ416342.1, update: 2008-10-23)DHA-1Positions 1-1113 of the gene (GenBank: EF406115.1, update: 2007-8-17). The result proves that the detection result by using the kit is accurate and reliable.
Figure IDA0000880191000000011
Figure IDA0000880191000000021
Figure IDA0000880191000000031
Figure IDA0000880191000000041
Figure IDA0000880191000000051
Figure IDA0000880191000000061
Figure IDA0000880191000000071
Figure IDA0000880191000000081
Figure IDA0000880191000000091
Figure IDA0000880191000000101
Figure IDA0000880191000000111

Claims (8)

1. The complete single-stranded DNA for detecting the drug resistance gene of the bacteria consists of a probe set and a primer pair set;
the primer pair group consists of 13 primer pairs as follows: a primer pair 1 consisting of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table; a primer pair 2 consisting of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table; a primer pair 3 consisting of two single-stranded DNA molecules shown in a sequence 5 and a sequence 6 in a sequence table; a primer pair 4 consisting of two single-stranded DNA molecules shown in a sequence 7 and a sequence 8 in a sequence table; a primer pair 5 consisting of two single-stranded DNA molecules shown in a sequence 9 and a sequence 10 in a sequence table; a primer pair 6 consisting of two single-stranded DNA molecules shown in a sequence 11 and a sequence 12 in a sequence table; a primer pair 7 consisting of two single-stranded DNA molecules shown in a sequence 13 and a sequence 14 in a sequence table; a primer pair 8 consisting of two single-stranded DNA molecules shown as a sequence 15 and a sequence 16 in a sequence table; a primer pair 9 consisting of two single-stranded DNA molecules shown in a sequence 17 and a sequence 18 in a sequence table; a primer pair 10 consisting of two single-stranded DNA molecules shown as a sequence 19 and a sequence 20 in a sequence table; a primer pair 11 consisting of two single-stranded DNA molecules shown in a sequence 21 and a sequence 22 in a sequence table; a primer pair 12 consisting of two single-stranded DNA molecules shown in a sequence 23 and a sequence 24 in a sequence table; a primer pair 13 consisting of two single-stranded DNA molecules shown in a sequence 25 and a sequence 26 in a sequence table;
the probe set consists of 13 single-stranded DNA probes as follows: a single-stranded DNA probe 1 shown as a sequence 27 in a sequence table; a single-stranded DNA probe 2 shown as a sequence 28 in a sequence table; a single-stranded DNA probe 3 shown as a sequence 29 in a sequence table; a single-stranded DNA probe 4 shown as a sequence 30 in the sequence table; a single-stranded DNA probe 5 shown as a sequence 31 in a sequence table; a single-stranded DNA probe 6 shown as a sequence 32 in the sequence table; a single-stranded DNA probe 7 shown as a sequence 33 in the sequence table; a single-stranded DNA probe 8 shown in a sequence 34 in a sequence table; a single-stranded DNA probe 9 shown in a sequence 35 in a sequence table; a single-stranded DNA probe 10 shown as a sequence 36 in a sequence table; a single-stranded DNA probe 11 shown in a sequence 37 in a sequence table; a single-stranded DNA probe 12 shown in a sequence 38 in the sequence table; a single-stranded DNA probe 13 shown as a sequence 39 in the sequence table.
2. The kit for detecting the drug-resistant gene of the bacteria comprises a primer pair group and a hybridization chip; the hybridization chip is prepared according to a method comprising the following steps: the general formula is' NH2-(T)n13 single-stranded detection probes of the hybridization probe sequence "were immobilized on aldehyde-modified solid-phase support by reaction of amino group with aldehyde group, respectively, to obtain the hybrid coreSlicing; (T) in the formulanN is an integer of 5 to 30 inclusive; the 13 hybridization probe sequences corresponding to the 13 single-stranded detection probes in the general formula are respectively shown as sequences 27-39 in a sequence table;
the primer pair group consists of 13 primer pairs as follows: a primer pair 1 consisting of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table; a primer pair 2 consisting of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table; a primer pair 3 consisting of two single-stranded DNA molecules shown in a sequence 5 and a sequence 6 in a sequence table; a primer pair 4 consisting of two single-stranded DNA molecules shown in a sequence 7 and a sequence 8 in a sequence table; a primer pair 5 consisting of two single-stranded DNA molecules shown in a sequence 9 and a sequence 10 in a sequence table; a primer pair 6 consisting of two single-stranded DNA molecules shown in a sequence 11 and a sequence 12 in a sequence table; a primer pair 7 consisting of two single-stranded DNA molecules shown in a sequence 13 and a sequence 14 in a sequence table; a primer pair 8 consisting of two single-stranded DNA molecules shown as a sequence 15 and a sequence 16 in a sequence table; a primer pair 9 consisting of two single-stranded DNA molecules shown in a sequence 17 and a sequence 18 in a sequence table; a primer pair 10 consisting of two single-stranded DNA molecules shown as a sequence 19 and a sequence 20 in a sequence table; a primer pair 11 consisting of two single-stranded DNA molecules shown in a sequence 21 and a sequence 22 in a sequence table; a primer pair 12 consisting of two single-stranded DNA molecules shown in a sequence 23 and a sequence 24 in a sequence table; and the primer pair 13 consists of two single-stranded DNA molecules shown in a sequence 25 and a sequence 26 in a sequence table.
3. The kit of claim 2, wherein: the kit also contains a random primer marked by fluorescence; the sequence of the random primer is 5' -NX-3', N represents any of A, G, C and T, 6 ≦ X ≦ 15, and X is an integer, NXRepresents X consecutive deoxyribonucleotides.
4. The kit of claim 2, wherein: the kit also contains a hybridization solution; the hybridization solution contains fluorescence-labeled unrelated single-stranded DNA molecules; the unrelated single-stranded DNA molecule is a single-stranded DNA molecule which is not derived from the drug resistance gene of the bacteria.
5. The kit of claim 4, wherein: the preparation method of the hybrid chip also comprises the step of fixing the surface chemical control probe QC, the hybrid quality control probe PC and the negative control probe BC on the aldehyde modified solid phase carrier through the reaction of amino and aldehyde;
the surface chemical quality control probe QC, the hybridization quality control probe PC and the negative control probe are single-stranded probes;
one end of the surface chemical quality control probe QC is modified by amino, and the other end of the surface chemical quality control probe QC is provided with a fluorescent label;
one end of the hybridization quality control probe PC is modified by amino and can be hybridized with the unrelated single-stranded DNA molecules in the hybridization solution;
one end of the negative control probe BC is modified with an amino group and is not hybridizable to any single-stranded DNA molecule derived from the bacterial drug resistance gene.
6. The kit of claim 5, wherein: the structural composition of the surface chemical quality control probe QC from 5 ' end to 3 ' end is ' NH2-TCACTTGCTTCCGTTGAGG-Hex”；
The structural composition of the hybridization quality control probe PC from the 5 ' end to the 3 ' end is ' NH2-TTTTTTTTTTTTCCTCAACGGAAGCAAGTGAT”；
The negative control probe BC has a structural composition from 5 ' end to 3 ' end as ' NH2-TTTTTTTTTTTTGTTGCTTCTGGAATGAGTTTGCT”。
7. Use of a set of single stranded DNA as claimed in claim 1 or a kit as claimed in any one of claims 2 to 6 in any one of:
(a) detecting or assisting in detecting bacterial drug resistance genes;
(b) preparing a product for detecting or assisting in detecting the drug resistance gene of the bacteria;
(c) detecting or assisting in detecting bacteria containing bacterial drug resistance genes;
(d) preparing a product for detecting or assisting in detecting bacteria containing bacterial drug resistance genes;
wherein the applications shown in (a) and (c) are non-disease diagnosis and treatment applications.
8. Use of a set of single-stranded DNA according to claim 1 for the preparation of a kit according to any one of claims 2 to 6.
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