CN106884038A - A kind of gene chip kit for detecting gram-positive bacterium drug resistant gene - Google Patents

Abstract

The invention discloses a gene chip kit for detecting drug-resistant genes of Gram-positive bacteria. The kit provided by the present invention contains a primer pair group for detecting drug-resistant genes of Gram-positive bacteria, consisting of 3 primer pairs, and its sequence is sequence 1-6 in the sequence table; meanwhile, the kit also contains Contains a hybridization chip immobilized with three single-stranded DNA probes shown in sequences 7-9. The kit provided by the invention supports high-throughput, rapid and accurate detection of multiple drug resistance genes of Gram-positive bacteria, and screening of Chinese population can effectively perform accurate diagnosis, drug resistance traceability, drug resistance control, etc. The role, thereby reducing the use of antibiotics and the emergence of drug resistance.

Description

A gene chip kit for detecting drug-resistant genes of Gram-positive bacteria

technical field

The invention belongs to the technical field of nucleic acid amplification, and relates to a gene chip kit for detecting drug-resistant genes of Gram-positive bacteria.

Background technique

Detection of bacterial drug resistance genes has important clinical significance. In clinical diagnosis, there are defects such as large dosage of antibiotics, high starting point, and relatively random types, which lead to the emergence and prevalence of drug-resistant bacteria. At present, the detection of drug resistance genes is still relatively traditional. There are generally physiological and biochemical tests, serological tests, drug sensitivity tests, etc. followed by polymerase chain reaction (PCR) methods to amplify the corresponding genes, but the above detection methods take a long time, The method is cumbersome in operation, slow in reporting results, and low in throughput, and the strains are often identified first before follow-up tests such as drug susceptibility can be carried out accordingly, so it is difficult to meet the needs of clinical treatment. Therefore, the development of rapid and high-throughput molecular diagnostic technology detection can assist rapid diagnosis and guide timely and accurate medication.

Biochip technology is a high-tech that has rapidly developed and matured in the field of life sciences in recent years. It is mainly a micro-biochemical analysis system constructed on the surface of a solid-phase chip through micro-processing technology and microelectronics technology, which can realize the analysis of cells, proteins, Accurate, rapid, and large-scale information detection of , nucleic acid, and other biological components. The main features of biochips are high throughput, miniaturization and automation.

Contents of the invention

The first object of the present invention is to provide a primer pair set for detecting drug resistance genes of Gram-positive bacteria.

The primer pair set used to detect Gram-positive bacterial drug-resistant genes provided by the present invention consists of the following 3 primer pairs:

1) The primer pair shown in (a1) or (a2) below is designated as primer pair 1:

(a1) a pair of primers consisting of two single-stranded DNA molecules shown in Sequence 1 and Sequence 2 in the sequence listing;

(a2) consists of two single-stranded DNA molecules shown in the sequence obtained by substituting and/or deleting and/or adding one or several nucleotides to Sequence 1 and Sequence 2 in the sequence listing, and is the same as (a1 A pair of primers having the same function as the pair of primers described in );

2) The primer pair shown in (b1) or (b2) below is designated as primer pair 2:

(b1) a pair of primers consisting of two single-stranded DNA molecules shown in sequence 3 and sequence 4 in the sequence listing;

(b2) consists of two single-stranded DNA molecules shown in the sequence obtained by substituting and/or deleting and/or adding one or several nucleotides to Sequence 3 and Sequence 4 in the Sequence Listing, and is the same as (b1 A pair of primers having the same function as the pair of primers described in );

3) The primer pair shown in (c1) or (c2) below is designated as primer pair 3:

(c1) a primer pair consisting of two single-stranded DNA molecules shown in sequence 5 and sequence 6 in the sequence listing;

(c2) consists of two single-stranded DNA molecules shown in the sequence obtained by substituting and/or deleting and/or adding one or several nucleotides to sequence 5 and sequence 6 in the sequence listing, and is the same as (c1 A pair of primers that are functionally identical to the pair of primers described in ).

Wherein, the primer pair 1 is used to amplify the mecA gene; the primer pair 2 is used to amplify the vanA gene; and the primer pair 3 is used to amplify the vanB gene.

The second object of the present invention is to provide a set of single-stranded DNA for detecting drug resistance genes of Gram-positive bacteria.

The set of single-stranded DNA for detecting bacterial drug-resistant genes provided by the present invention is specifically composed of a probe set and the primer pair set; the probe set is composed of the following three single-stranded DNA probes: in the sequence listing Single-stranded DNA probe 1 shown in sequence 7; single-stranded DNA probe 2 shown in sequence 8 in the sequence listing; single-stranded DNA probe 3 shown in sequence 9 in the sequence listing.

Wherein, the single-stranded DNA probe 1 is used to detect the amplification result of the primer pair 1; the single-stranded DNA probe 2 is used to detect the amplification result of the primer pair 2; the single-stranded DNA probe Needle 3 is used to detect the amplification result of the primer pair 3.

The third object of the present invention is to provide a kit for detecting drug resistance genes of Gram-positive bacteria.

The kit for detecting drug-resistant genes of Gram-positive bacteria provided by the present invention contains 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 The three single-strand detection probes of "NH ₂ -(T) _n -hybridization probe sequence" are respectively immobilized on an aldehyde-modified solid phase carrier (such as a glass slide) through the reaction of an amino group and an aldehyde group, and the described Hybridization chip; (T) _n in the general formula represents n consecutive Ts, and n is an integer greater than or equal to 5 and less than or equal to 30; in the general formula, corresponding to the three single-stranded detection probes The sequences of the three kinds of hybridization probes are respectively shown as sequences 7-9 in the sequence listing.

According to needs, the kit can also contain fluorescently labeled random primers; the sequence of the random primers is 5'-N _X -3', N represents any one of A, G, C and T, 6 ≤X≤15, and X is an integer (such as X=9), N _X represents X consecutive deoxyribonucleotides.

The above fluorescently labeled random primers are used for fluorescently labeling the amplification products of the primer pairs in the primer pair group. According to needs, other methods may be used instead of fluorescently labeling the amplification product with the fluorescently labeled random primers. If the 5' end of one primer of each primer pair in the primer pair set is fluorescently labeled (such as TAMRA), the fluorescently labeled primer exists on the single-stranded DNA that can be hybridized by the corresponding probe in the amplification product primers.

The kit may also contain a hybridization solution; the hybridization solution contains fluorescently labeled irrelevant single-stranded DNA molecules; the irrelevant single-stranded DNA molecules are single-stranded DNA molecules not derived from the bacterial drug resistance gene .

The preparation method of the hybridization chip also includes fixing the surface chemical quality control probe QC, and/or the hybridization quality control probe PC, and/or the negative control probe BC to the modified aldehyde group through the reaction of the amino group and the aldehyde group. The steps on the solid phase carrier (such as glass slide) of liquefaction;

The surface chemical quality control probe QC, the hybridization quality control probe PC and the negative control probe are all single-stranded probes; one end of the surface chemical quality control probe QC is modified with an amino group, and the other end has Fluorescent label (such as Hex); one end of the hybridization quality control probe PC is modified with an amino group, and can hybridize with the irrelevant single-stranded DNA molecule in the hybridization solution; one end of the negative control probe BC is modified with an amino group Modified, and cannot hybridize to any single-stranded DNA molecule derived from the Gram-positive bacterial drug resistance gene.

Further, in the present invention, the structural composition of the surface chemical quality control probe QC from the 5' end to the 3' end is "NH ₂ -TCACTTGCTTCCGTTGAGG-Hex"; the hybridization quality control probe PC is from the 5' end to the 3' end The structural composition of the 'end is "NH ₂ -TTTTTTTTTTTTTCCTCAACGGAAGCAAGTGAT"; the structural composition of the negative control probe BC from the 5' end to the 3' end is "NH ₂ -TTTTTTTTTTTTGTTGCTTCTGGAATGAGTTTGCT".

The application of the primer pair group or the set of single-stranded DNA or the kit in the following (a) or (b) also belongs to the protection scope of the present invention:

(a) Detect or assist in the detection of Gram-positive bacterial drug resistance genes (non-diagnostic purposes), or prepare products for the detection or auxiliary detection of Gram-positive bacterial drug resistance genes;

(b) Detection or auxiliary detection of bacteria containing Gram-positive bacterial drug resistance genes (non-diagnostic purposes), or preparation of products for detection or auxiliary detection of bacteria containing Gram-positive bacterial drug resistance genes.

In the present invention, the Gram-positive bacterial drug resistance gene can specifically be at least one of the following: mecA gene, vanA gene, vanB 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 The accession number is AY655711.1 (update: 2005-11-7).

Correspondingly, the bacterium containing the mecA gene can specifically be Staphylococcus aureus; the bacterium containing the vanA gene can specifically be Enterococcus faecalis or Enterococcus faecium; the bacterium containing the vanB gene can specifically be Enterococcus faecalis or Enterococcus faecium.

The application of the primer pair group or the set of single-stranded DNA in the preparation of the kit also belongs to the protection scope of the present invention.

The invention studies the drug-resistant genes of Gram-positive bacteria with a very large sample size. The kit provided by the invention supports high-throughput, rapid and accurate detection of multiple drug-resistant genes of Gram-positive bacteria, and screening of the Chinese population can effectively perform accurate diagnosis, traceability of drug resistance, control of drug resistance, etc. The role, thereby reducing the use of antibiotics and the emergence of drug resistance.

Description of drawings

FIG. 1 is a schematic diagram of a dot array arrangement of a hybridization chip (ie, a schematic diagram of a chip probe layout). - indicates that no probe is fixed.

Fig. 2 is the specificity analysis result of the kit used to detect drug resistance genes of Gram-positive bacteria. The schematic diagram of chip probe layout corresponding to this figure is shown in FIG. 1 . Among them, the reference DNA plasmid corresponding to the chip detection result shown in A is ZL-vanA (corresponding to the vanA gene); the reference DNA plasmid corresponding to the chip detection result shown in B is ZL-vanB (corresponding to the vanB gene); the chip shown in C The reference DNA plasmid corresponding to the test result is ZL-mecA (corresponding to the mecA gene).

Fig. 3 is the detection result of the clinical sample of the kit for detecting the drug resistance gene of Gram-positive bacteria. The schematic diagram of chip probe layout corresponding to this figure is shown in FIG. 1 . Among them, the chip test result shown in A corresponds to clinical sample No. 1; the chip test result shown in B corresponds to clinical sample No. 2; the chip test result shown in C corresponds to clinical sample No. 3.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, preparation and use thereof for detecting the kit of Gram-positive bacterial drug resistance gene

1. Assembly and preparation of kits for detecting drug resistance genes of Gram-positive bacteria

1. Primers and hybridization probes designed for 3 drug-resistant genes of Gram-positive bacteria

The specific sequences of primers and single-stranded hybridization probes designed for the three bacterial drug resistance genes are shown in Table 1 and Table 2.

Table 1 Primers designed for 3 drug resistance genes of Gram-positive bacteria

Table 2 Single-stranded hybridization probes designed for three Gram-positive bacterial drug resistance genes

serial number detection gene GenBank probe name Sequence (5'-3') 1 mecA AB221119.1 mecA-1427 GTAGCACTCGAATTAGGCAG (Sequence 7) 2 van A M97297.1 vanA-379 TGTAGGCTGCGATATTCAAA (sequence 8) 3 van B AY655711.1 vanB-793 GTCGAGGAACGAAATCGGGT (sequence 9)

2. Immobilize the hybridization probe on the hybridization chip

The hybridization chip is a substrate on which three kinds of single-stranded detection probes are immobilized respectively. Each detection probe is an amino-modified oligonucleotide probe. The general formula of the three single-stranded detection probes is "NH ₂ -TTTTTTTTTTTTTTTT-hybridization probe sequence", wherein the "hybridization probe sequence" is Three kinds of single-stranded hybridization probes shown in the sequences 7-9 in Table 2". Each detection probe was dissolved with gene spotting solution (product of Boao Biological Group Co., Ltd., its product catalog number is CP.440010) respectively, and finally The concentration was 10μM, and it was spotted three times onto the aldehyde-modified glass slide (product of Boao Biological Group Co., Ltd., its product catalog number is CP.420022), so that the three probes were immobilized through the reaction of amino groups and aldehyde groups. On the hybridization chip.In addition, the surface chemical quality control probe QC, the hybridization quality control probe PC and the negative control probe BC are also fixed on the hybridization chip by the reaction of amino and aldehyde groups.QC is that one end has a Hex label , the other end has an amino-modified single-stranded oligonucleotide probe, which is used to observe the efficiency of chip spotting and immobilization. Its structural composition from the 5' end to the 3' end is NH ₂ -TCACTTGCTTCCGTTGAGG-Hex. PC is an amino group Modified single-stranded oligonucleotide probes, which can be hybridized with fluorescently labeled irrelevant single-stranded DNA molecules (C-PC) added to the hybridization solution, used for quality control of the hybridization process, from the 5' end to the 3' The structural composition of the end is NH ₂ -TTTTTTTTTTTTTCCTCAACGGAAGCAAGTGAT. BC is an amino-modified oligonucleotide probe that will not hybridize to all sequences to be detected in the hybridization system and is used to observe non-specific hybridization. It extends from the 5' end to The structural composition of the 3' end is NH ₂ -TTTTTTTTTTTTGTTGCTTCTGGAATGAGTTTGCT. Figure 1 is a schematic diagram of the array arrangement of the hybridization chip.

3. The composition of the kit for detecting drug-resistant genes of Gram-positive bacteria

The kit provided by the present invention for detecting drug-resistant genes of Gram-positive bacteria (i.e. 3 kinds of drug-resistant genes of Gram-positive bacteria involved in Table 1 and Table 2) contains:

(1) 3 primer pairs shown in Table 1 in step 1;

(2) In step 2, 3 kinds of detection probes, surface chemical quality control probe QC, hybridization quality control probe PC and negative control probe BC are immobilized on the hybridization chip;

(3) Fluorescently labeled random primers; the sequence of the random primers is 5'-N ₉ -3', N represents any one of A, G, C and T, and N ₉ represents 9 consecutive deoxyribose sugars Nucleotides.

(4) Hybridization solution; the hybridization solution contains a fluorescently labeled irrelevant single-stranded DNA molecule (C-PC), whose nucleotide sequence is 5'-ATCACTTGCTTCCGTTGAGG-3'.

2. The method of using the kit for detecting drug resistance genes of Gram-positive bacteria

1. Sample polymerase chain reaction (PCR) amplification

Take 1 μL of the DNA sample solution to be tested, use it as a template, and perform PCR amplification using the three primer pairs shown in Table 1, respectively. The 20 μL PCR amplification system is as follows: 10×PCR buffer (containing Mg ²⁺ ) 2 μL; 2.5 mM dNTP 1.6 μL; 10 μM upstream primer 0.5 μL; 10 μM downstream primer 0.5 μL; template 1 μL; 5U/μL rTaq 0.2 μL; The PCR amplification program was as follows: 94°C for 5min; (94°C for 30s; 55°C for 30s; 72°C for 1min) 30 cycles; 72°C for 10min. After the PCR reaction, a total of 3 PCR amplification products were obtained.

2. Fluorescent labeling of PCR amplification products

5 μL of each PCR amplification product was added to different sample tubes, and 3 μL of 100 μM TAMRA fluorescently labeled 9N random primers (nucleotide sequence 5′-N9-3′, N Indicates any one of A, G, C, and T, N9 indicates 9 consecutive deoxyribonucleotides, specifically the product of Sangon Bioengineering (Shanghai) Co., Ltd.), add water to 19 μL, shake and mix, and instantly Centrifuge; place on ice after denaturation at 95°C, add 6 μL fluorescent labeling reaction system mix (composition: 10×Klenow Buffer 2.5 μL; 5 U/μL Klenow enzyme 1 μL; 2.5 mM dNTP 2.5 μL). Fluorescent labeling was carried out according to the program, the program was as follows: 90 min at 37°C; 10 min at 70°C. A total of 3 TAMRA fluorescently labeled products were obtained.

3. Purification of TAMRA fluorescently labeled products

Use the Nucleo Spin Gel and PCR Clean-up kit produced by Macherey-Nagel (product number: REF740609*250) to purify the TAMRA fluorescently labeled product obtained in step 2, and the specific operation was performed according to the kit instructions.

4. Hybridization

Melt the hybridization buffer containing unrelated single-stranded DNA molecules (5'-ATCACTTGCTTCCGTTGAGG-3') labeled with TAMRA fluorescence (product of Boao Biological Group Co., Ltd., its catalog number is CP.440030) at 50°C, and prepare 3 parts Hybridization mixture (including 15 μL of the purified TAMRA fluorescent labeling product solution in step 3, and 5 μL of hybridization buffer), add the prepared 3 hybridization mixtures to the hybridization chip in step 1 and 2, and each hybridization mixture corresponds to a complete The chip was hybridized, denatured at 95°C for 3 minutes, immediately placed on ice, and hybridized in a water bath at 50°C for 2 hours.

5. Cleaning

Use two different washing solutions (washing solution I: 2×SSC, 0.2% SDS, preheated to 50°C; washing solution II: 0.2×SSC, preheated to 50°C) in the order of first washing solution I and then washing solution II After washing for 4 minutes respectively, put the chip in the SlideWasher_8 chip washer, select the centrifugation program, centrifuge (or centrifuge at 1000 rpm for 2 minutes), and spin dry.

6. Scanning and result judgment

Use Boao Jingxin LuxScan 10K microarray chip scanner to complete the scan (select the green channel, set the parameter range of "Power" to 50-90 and the parameter range of "PMT" to 500-900), and according to the scanning results as follows Methods Determine whether the DNA sample to be tested contains the 3 Gram-positive bacterial drug-resistant genes involved in Table 1, and which one or several of the 3 Gram-positive bacterial drug-resistant genes are specifically contained: for If a fluorescent signal is detected at a fixed position on the chip by the probe for detecting a Gram-positive bacterial drug-resistant gene, it is determined that the corresponding DNA sample to be tested contains the corresponding Gram-positive bacterial drug-resistant gene; otherwise, no Contains the corresponding Gram-positive bacterial resistance genes. In addition, during the scanning process, the software will give different colors according to the intensity of the detected fluorescent signal, the color is from blue to white, and the signal will gradually increase, so as to preliminarily judge the level of drug resistance gene content of target Gram-positive bacteria in the DNA sample to be tested .

Embodiment 2, the specificity and the sensitivity determination of the kit for detecting drug resistance gene of Gram-positive bacteria

1. Preparation of reference DNA plasmid

1. Construction of a plasmid containing the mecA gene target fragment

The DNA fragment shown at positions 1001-1926 of the mecA gene (GenBank: AB221119.1, update: 2006-5-19) was ligated to the pGEM-T Easy Vector vector (promega company product) to obtain the recombinant plasmid ZL-mecA. and verified by sequencing.

2. Construction of a plasmid containing the vanA gene target fragment

The DNA fragment shown at position 7000-7988 of the vanA gene (GenBank: M97297.1, update: 2002-6-20) was ligated to the pGEM-T Easy Vector vector to obtain the recombinant plasmid ZL-vanA. and verified by sequencing.

3. Construction of plasmids containing vanB gene target fragments

The DNA fragment shown at positions 14-1029 of the vanB gene (GenBank: AY655711.1, update: 2005-11-7) was ligated to the pGEM-T Easy Vector vector to obtain the recombinant plasmid ZL-vanB. and verified by sequencing.

2. Specificity analysis of kits for detecting drug resistance genes of Gram-positive bacteria

Dilute the three kinds of reference product DNA plasmids constructed in step 1 to a concentration of 10 ⁴ copies/μL, and use the diluted three kinds of reference product DNA plasmids as the DNA samples to be tested, and then perform the operation according to step 2 of Example 1 to detect Whether the DNA sample to be tested contains the drug-resistant gene of the target Gram-positive bacteria, the specific determination method can be found in Step 2 6 of Example 1.

The results are shown in Figure 2. It can be seen from the figure that for each reference product DNA plasmid, only the spots where the corresponding detection probes are immobilized can detect obvious fluorescent signals, and no fluorescent signals can be detected at other spots. This result shows that the kit for detecting drug-resistant genes of Gram-positive bacteria provided by the present invention has strong specificity.

3. Sensitivity analysis of kits used to detect drug resistance genes of Gram-positive bacteria

The three reference DNA plasmids constructed in the above step 1 were serially diluted to obtain dilutions with concentrations of 10 ⁴ copies/μL, 10 ³ copies/μL, 10 ² copies/μL, and 10 ¹ copies/μL. Three kinds of reference DNA plasmids with different dilutions were used as the DNA samples to be tested, and then operated according to step 2 of Example 1 to detect whether the DNA samples to be tested contained the target Gram-positive bacterial drug resistance gene. For specific determination methods, see Embodiment 1 step two 6.

The results show that: for each reference product DNA plasmid, when the concentration is 10 ⁴ copies/μL, 10 ³ copies/μL, and 10 ² copies/μL, the spots of the corresponding detection probes can detect obvious fluorescent signals; No fluorescent signal was detected for the reference DNA plasmid at 10 ¹ copies/μL. The result shows that the kit provided by the present invention for detecting drug-resistant genes of Gram-positive bacteria has high sensitivity.

Embodiment 3, the detection of actual clinical sample

1. Types of clinical samples

The clinical samples used in this example came from viscous pus from human wounds collected by Peking University People's Hospital (based on the principle of voluntariness of the collectors), a total of three samples.

2. Extraction of DNA samples from clinical samples

1. Take 1-3mL of clinical samples from step 1;

2. Add 4 times the volume of 4% (4g/100mL) NaOH, shake well, and place at room temperature for 30 minutes to liquefy;

3. Take 0.5ml of liquefied pus and 0.5mL of 4% (4g/100mL) NaOH, room temperature, 10min;

4. Centrifuge at 12 000 rpm for 15 minutes;

5. Discard the supernatant, add 1 mL of sterile saline, mix well, and centrifuge at 12 000 rpm for 5 min;

6. Discard the supernatant, and precipitate for DNA extraction;

7. Add 50 μL of nucleic acid extraction solution (product of Boao Biological Group Co., Ltd., its catalog number is CP.360090), shake and mix well to completely suspend the precipitate;

8. Transfer the suspension to a nucleic acid extraction tube, tighten the cap, and put it into a nucleic acid extraction instrument or vortex shaker for 5 minutes at the maximum speed;

9. Heating in a metal bath at 95°C for 5 minutes;

10. Centrifuge at 5000rpm for 1min, transfer the supernatant to a 1.5mL centrifuge tube, and store at -20°C for later use.

3. Detection of actual clinical samples

The DNA of the three clinical samples obtained in step 2 is used as the DNA sample to be tested, and then the operation is carried out according to step 2 of Example 1 to detect whether the DNA sample to be tested contains the drug resistance gene of the target Gram-positive bacteria. For the specific determination method, see the example 1 step two 6.

The results are shown in Figure 3, from which it can be seen that the hybridization signals of the three samples are clear and single. After comparison with the position of the probe, the signal detected in the No. 1 clinical sample is the drug-resistant gene mecA, the signal detected in the No. 2 clinical sample is the drug-resistant gene mecA and the drug-resistant gene vanA, and the signal detected in the No. 3 clinical sample is Drug resistance gene vanB.

In order to further confirm the accuracy of the above 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 used the primer pair mecA -The amplification product of F/mecA-R has obvious electrophoretic bands, while the amplification products of other primer pairs have no obvious electrophoretic bands. Further, the amplification products of the primer pair mecA-F/mecA-R Sequencing found that its sequence was the 1001-1926th position of the mecA gene (GenBank: AB221119.1, update: 2006-5-19); No. 2 clinical sample only used the primer pair mecA-F/mecA-R and vanA-F/ The amplified products of vanA-R had obvious electrophoretic bands, while the amplified products of other primer pairs had no obvious electrophoretic bands. Further primer pairs mecA-F/mecA-R and vanA-F/vanA- The sequence of the amplified product of R was found to be the 1001-1926th position of the mecA gene (GenBank: AB221119.1, update: 2006-5-19) and the vanA gene (GenBank: M97297.1, update: 2002-6- No. 7000-7988 of 20); clinical sample No. 3 had obvious electrophoresis bands only in the amplified products of the primer pair vanB-F/vanB-R, while no obvious electrophoresis was detected in the amplified products of other primer pairs Further sequencing of the amplified product of the primer pair vanB-F/vanB-R found that its sequence was the 14th-1029th position of the vanB gene (GenBank: AY655711.1, update: 2005-11-7). This result proves that the detection result using the kit of the present invention is accurate and reliable.

Claims (10)

1. A primer pair set for detecting drug-resistant genes of Gram-positive bacteria, consisting of the following 3 primer pairs: 1) The primer pair shown in (a1) or (a2) below is designated as primer pair 1: (a1) a pair of primers consisting of two single-stranded DNA molecules shown in Sequence 1 and Sequence 2 in the sequence listing; (a2) consists of two single-stranded DNA molecules shown in the sequence obtained by substituting and/or deleting and/or adding one or several nucleotides to Sequence 1 and Sequence 2 in the sequence listing, and is the same as (a1 A pair of primers having the same function as the pair of primers described in ); 2) The primer pair shown in (b1) or (b2) below is designated as primer pair 2: (b1) a pair of primers consisting of two single-stranded DNA molecules shown in sequence 3 and sequence 4 in the sequence listing; (b2) consists of two single-stranded DNA molecules shown in the sequence obtained by substituting and/or deleting and/or adding one or several nucleotides to Sequence 3 and Sequence 4 in the Sequence Listing, and is the same as (b1 A pair of primers having the same function as the pair of primers described in ); 3) The primer pair shown in (c1) or (c2) below is designated as primer pair 3: (c1) a primer pair consisting of two single-stranded DNA molecules shown in sequence 5 and sequence 6 in the sequence listing; (c2) consists of two single-stranded DNA molecules shown in the sequence obtained by substituting and/or deleting and/or adding one or several nucleotides to sequence 5 and sequence 6 in the sequence listing, and is the same as (c1 A pair of primers that are functionally identical to the pair of primers described in ). 2. be used for detecting the complete single-stranded DNA of Gram-positive bacterial drug-resistant gene, be made up of probe group and the primer pair group described in claim 1; The probe set is composed of the following three single-stranded DNA probes: single-stranded DNA probe 1 shown in sequence 7 in the sequence listing; single-stranded DNA probe 2 shown in sequence 8 in the sequence listing; 9 shows ssDNA probe 3. 3. the test kit that is used to detect Gram-positive bacterial drug resistance gene, contains the primer pair group described in claim 1, and hybridization chip; The hybridization chip is prepared according to the following steps: three single-stranded detection probes with the general formula "NH ₂ -(T) _n -hybridization probe sequence" are immobilized through the reaction of amino groups and aldehyde groups respectively On the aldehyde-modified solid phase carrier, obtain the hybridization chip; (T) _n in the general formula represents n continuous Ts, and n is an integer greater than or equal to 5 and less than or equal to 30; the general formula The sequences of the three hybridization probes corresponding to the three single-stranded detection probes in the formula are respectively shown in sequences 7-9 in the sequence listing. 4. The test kit according to claim 3, characterized in that: the kit also contains fluorescently labeled random primers; the sequence of the random primers is 5'-N _X -3', N represents A, Any one of G, C and T, 6≤X≤15, and X is an integer, and N _X represents X consecutive deoxyribonucleotides. 5. The kit according to claim 3 or 4, characterized in that: said kit also contains a hybridization solution; said hybridization solution contains fluorescently labeled irrelevant single-stranded DNA molecules; said irrelevant single-stranded DNA Molecules are single-stranded DNA molecules not derived from the Gram-positive bacterial resistance genes. 6. kit according to claim 5, is characterized in that: the preparation method of described hybridization chip also comprises surface chemical quality control probe QC and/or hybridization quality control probe PC and/or negative control probe The step of immobilizing BC on the aldehyde-modified solid phase support through the reaction of amino group and aldehyde group; The surface chemical quality control probe QC, the hybridization quality control probe PC and the negative control probe are all single-stranded probes; One end of the surface chemical quality control probe QC is modified with an amino group, and the other end has a fluorescent label; One end of the hybridization quality control probe PC is modified with an amino group, and can hybridize with the irrelevant single-stranded DNA molecule in the hybridization solution; One end of the negative control probe BC is modified with an amino group, and cannot hybridize with any single-stranded DNA molecule derived from the bacterial drug resistance gene. 7. The kit according to claim 6, characterized in that: the structural composition of the surface chemical quality control probe QC from the 5' end to the 3' end is "NH ₂ -TCACTTGCTTCCGTTGAGG-Hex"; The structural composition of the hybridization quality control probe PC from the 5' end to the 3' end is "NH ₂ -TTTTTTTTTTTTTCCTCAACGGAAGCAAGTGAT"; The structural composition of the negative control probe BC from the 5' end to the 3' end is "NH ₂ -TTTTTTTTTTTTGTTGCTTCTGGAATGAGTTTGCT". 8. the application of the primer pair group described in claim 1 or the complete set of single-stranded DNA described in claim 2 or the kit described in any one of claims 3-7 in the following (a) or (b): (a) Detect or assist in the detection of drug resistance genes of Gram-positive bacteria, or prepare products for the detection or auxiliary detection of drug resistance genes of Gram-positive bacteria; (b) Detection or auxiliary detection of bacteria containing Gram-positive bacterial drug resistance genes, or preparation of products for detection or auxiliary detection of bacteria containing Gram-positive bacterial drug resistance genes. 9. According to any one of claims 1-8, the primer pair group or complete set of single-stranded DNA or kit or application is characterized in that: the drug-resistant gene of Gram-positive bacteria is at least one of the following: mecA gene, vanA gene, vanB gene. 10. Use of the primer pair set according to claim 1 or the set of single-stranded DNA according to claim 2 in the preparation of the kit according to any one of claims 3-7.