CN108642169A - Detect ten kinds of trimethoprim class Drug-resistant genetic methods and used kit - Google Patents

Detect ten kinds of trimethoprim class Drug-resistant genetic methods and used kit Download PDF

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CN108642169A
CN108642169A CN201810352337.7A CN201810352337A CN108642169A CN 108642169 A CN108642169 A CN 108642169A CN 201810352337 A CN201810352337 A CN 201810352337A CN 108642169 A CN108642169 A CN 108642169A
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class drug
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trimethoprim
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张琼
李兰娟
项春生
刁宏燕
刘芳
孟晓华
姚坚
周佳维
吴灵娇
倪淑君
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Zhejiang University ZJU
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Abstract

The invention discloses a kind of kits of 10 kinds of trimethoprim class Drug-resistant genes of detection, it includes the amplimer combination that 10 kinds of trimethoprim class Drug-resistant genes are carried out with specific amplification, further includes the specific probe for detecting 10 kinds of different trimethoprim class Drug-resistant genes.The method that the present invention further simultaneously discloses the 10 kinds of trimethoprim class Drug-resistant genes of detection carried out using the kit.The method and its kit of detection trimethoprim class Drug-resistant gene of the present invention, it is good with integration degree height, high sensitivity, specificity, testing result is reliable and stable, feature at low cost, it can be widely used for distribution, the epidemiological surveillance of trimethoprim class Drug-resistant gene, for the rational use of medicines, effectively reduces and drug resistant generation is delayed to provide effective tool.

Description

Detect ten kinds of trimethoprim class Drug-resistant genetic methods and used kit
Technical field
The present invention relates to the methods and its kit of a kind of 10 kinds of trimethoprim class Drug-resistant genes of detection.
Background technology
One, trimethoprim class drug and its mechanism of action：
Trimethoprim (Trimethoprin, TMP) is a kind of synthetic antibacterial agents.The first TMP compound 1962 It is used for the first time in Britain.Since the combination of TMP-SUL is shown in vitro experiment with synergistic effect, since nineteen sixty-eight, TMP often with sulfanilamide (SN) (Sulfonamide, SUL) class Drug combination.However, clinical experience shows TMP-SUL combinations in body It is interior that there is no apparent synergistic effects.TMP is used alone since the 1970s, is initially used for preventing urinary tract infections, so After be used for treat acute urinary tract infection.
TMP can be with the dihyrofolate reductase (DHFR in bacterium；EC 1.5.1.3) it combines, inhibit dihydrofolate reduction Enzyme makes dihydrofolate reduction be obstructed at tetrahydrofolic acid.Tetrahydrofolic acid is necessary for folic acid synthesis, and folic acid is to form core The important as precursors of thuja acid and amino acid.TMP is by inhibiting tetrahydrofolic acid formation to reach antibacterial purpose.
TMP has extensive antimicrobial spectrum, including Escherichia coli (Escherichia coli) and enterobacteriaceae (Enterobacteriaceae) other members, streptococcus pneumonia (Streptococcus pneumoniae), the bloodthirsty bar of influenza Bacterium (Haemophilus influenzae), moraxelle catarrhalis (Moraxella catarrhalis), staphylococcus aureus (Staphylococcus aureus), Shigella (Shigella sp.) etc..These pathogens can cause urethra, breathing Road, intestines problem and skin disease.
Since clinical indication is extensive, TMP class drugs are widely used to all over the world.Further, since this kind of synthesisization It is relatively cheap to close price lattice so that TMP is widely used in developing country and some relatively poor countries.It is reported that hair The TMP resistant rates of national gram-negative pathogens are apparently higher than developed country, South America, Asia and Africa, TMP resistances in exhibition It is in 25% to 68% high level.Meanwhile developing country shows that the excrement of very high TMP drug resistance enterobacterias carries water It is flat.The increase of some drug taking behaviors lack of standardization in expanding and use with the range used, to the drug resistance of TMP class drugs International extensive attention is caused.
Two, resistance mechanism of the bacterium to trimethoprim class drug：
The resistance that bacterium can be divided into chromosome resistance to the resistance mechanism of TMP and plasmid carries.Plasmid-mediated TMP resistances It was found first in 1972, Pattishall et al. has found the DHFR for mediating the plasmid of TMP resistances to carry (relative to chromosome The special DHFR of coding, referred to as assists DHFR, by dfr gene codes).TMP resistant genes use complicated metastasis, including The horizontal heredity of locus specificity recombination exchanges, and TMP resistant genes are propagated in various pathogenic bacteria.Tn7 transposon, is inserted integron The miniplasmids for entering the factor (IS) and broad host range is the important carrier for carrying and shifting auxiliary DHFR.
Drug resistance database (The Comprehensive Antibiotic Resistance are integrated according to antibiotic Database, CARD) analysis and arrangement, 30 kinds of different auxiliary dfr gene types are had discovered that in different bacterium at present, Include 20 kinds of dfrA genes and 10 kinds of non-dfrA classes TMP drug resistant genes (dfrB1, dfrB2, dfrB3, dfrB6, dfrC, DfrD, dfrE, dfrF, dfrG and dfrK), wherein dfrB genoids are distributed in pseudomonad, Klebsiella and Salmonella more In bacterium and some Gram-negative bacterias that can not yet cultivate, dfrC-dfrG type TMP drug resistant genes more be distributed in staphylococcus, In the gram-positive bacterias such as enterococcus, listeria spp and streptococcus.
Three, the Drug Resistance Detection method of trimethoprim class drug：
Bacterium has two classes, i.e. Phenotypic examination and genotype to the detection method of trimethoprim class drug resistance at present Detection.After Phenotypic examination is by isolating and purifying the pathogen in sample, cause of disease is cultivated again in the presence of TMP drugs Bacterium by inhibition zone size, or measures semilethal drug concentration (MIC50) carry out；And genotype detection then passes through molecular biology Whether method, detection bacterium carry and the relevant gene of trimethoprim class drug resistance.
Phenotypic examination step is complicated, and time-consuming, and whole process at least needs 48-72 hours, in pathogenicbacteria separation incubation, Due to there is no antibiotic selective pressure, the TMP resistant genes of the carryings such as plasmid may be made to lose, cause subsequent drug quick Perception detection obtains the result of false negative.The advantages of genotype detection is the drug resistance information that can provide gene level, can be in sample It is obtained in 24 hours as a result, technical step is less after collection, expense is relatively inexpensive under normal circumstances.
The result and genotype call results of phenotypic resistance detection are usually identical but sometimes also variant.And largely face Bed practice have shown that, if genotype detection arrive drug resistant gene, even if Phenotypic examination be shown as sensitivity, still there is a strong possibility is used The result of medicine failure.This phenomenon is the false negative result obtained due to Phenotypic examination, and actually bacterium carries drug resistant gene, Dominant strain is quickly become under medicament selection pressure so that medication fails after medication.In view of this, more and more in the world Bacterial drug resistance researcher and clinician suggest reference gene type testing result, and genotype detection is to related drug resistant gene, i.e., It is sensitivity to make drug sensitive test experimental result, still it is not recommended that using corresponding antibiotic, to delay and weaken bacterium as far as possible to antibiosis The drug resistant occurrence and development of element.
Although genotype detection result is accurate, it can only detect the drug resistant gene of known array, and to not finding Novel drug resistant gene, due to gene order is unknown and can not targeted design specific primer and probe and can not detect.With This is on the contrary, Phenotypic examination due to directly detecting survival state of the bacterial strain under medicament selection pressure, thus is directed to novel drug resistance base The drug resistance caused by is it is possible to obtain positive test symbol.Therefore, there is still a need for combine Phenotypic examination as a result, just for genotype detection It can carry out comprehensive descision.
Invention content
Good, high sensitivity that the technical problem to be solved in the present invention is to provide a species specificity, detection 10 quick, at low cost The method and its kit of kind trimethoprim class Drug-resistant gene (table 1).
In order to solve the above technical problem, the present invention provides a kind of 10 kinds of trimethoprim class Drug-resistant genes of detection Kit：
Include the amplimer combination that 10 kinds of trimethoprim class Drug-resistant genes are carried out with specific amplification, sequence Row such as SEQ ID NO:Shown in 1-20；
Further include the specific probe for detecting 10 kinds of different trimethoprim class Drug-resistant genes, sequence such as SEQ ID NO:Shown in 21-30.
The improvement of the kit of 10 kinds of trimethoprim class Drug-resistant genes of detection as the present invention：Specificity is visited Needle is fixed on carrier；The carrier is following any：
Silicon chip, the film derived from various functions group, with slide carrier (the preferably aldehyde radical base of various functions base group modification The slide of group's modification).
The kit of 10 kinds of trimethoprim class Drug-resistant genes of detection as the present invention is further improved：Institute It further includes pcr amplification reaction liquid and hybridization reaction solution (that is, the required reaction solution of PCR amplification, hybridization reaction) to state kit.
The present invention is gone back while providing the 10 kinds of trimethoprim class Drug-resistants of detection carried out using mentioned reagent box The method of gene, includes the following steps：
1) the amplimer combination for, including using kit and pcr amplification reaction liquid, to that may be present in sample to be tested Included at least one of 10 kinds of trimethoprim class Drug-resistant genes to be measured (any type and several gene regions Section) asymmetric amplification is carried out, while fluorescent marker is carried out to PCR product；
2), the amplified production obtained by step 1) is mixed with the hybridization reaction solution in kit, then with specific probe into Row hybridization；Scanned (scanner can be determined according to the carrier of fixed specific probe and the type of PCR product label) obtains every Signal value, the background value (being gray value) of a specific probe；Calculate the signal-to-noise ratio of every specific probe：Signal-to-noise ratio= (signal value-background value)/background value；When signal-to-noise ratio is more than or equal to 3, judge that the genetic test result corresponding to the probe is sun Property, such as all specific probe signal-to-noise ratio are respectively less than 3, then judge phonetic not comprising 10 kinds of emilium tosylates to be measured in the sample to be tested Pyridine class Drug-resistant gene.
The improvement of the method for 10 kinds of trimethoprim class Drug-resistant genes of detection as the present invention：To be measured 10 kinds Trimethoprim class Drug-resistant gene is：dfrB1、dfrB2、dfrB3、dfrB6、dfrC、dfrD、dfrE、dfrF、dfrG And dfrK.Shown in table 1 specific as follows.
The drug resistant gene of 1. 10 kinds of table trimethoprim class drug to be detected
The kit of the present invention includes that 10 kinds of drug resistant gene sections are carried out with the primer combination of specific amplification；With above-mentioned expansion The PCR product of increasing is hybridized, and the specific probe and assist probes of different dfr drug resistant genes are detected；And PCR amplification and miscellaneous It hands over and reacts required reaction solution.The method of the present invention includes multiple asymmetric PCR amplification method and specific hybrid sides Method.
There is the specific primer of the trimethoprim class Drug-resistant gene dfrB1 sequence 1 in sequence table to arrive The nucleotide sequence of sequence 2, specific probe have the nucleotide sequence of sequence 21 in sequence table；The trimethoprim The specific primer of class Drug-resistant gene dfrB2 arrives the nucleotide sequence of sequence 4 with sequence 3 in sequence table, and specificity is visited The nucleotide sequence of sequence 22 in needle set ordered list；The trimethoprim class Drug-resistant gene dfrB3's is special Property primer have sequence table in sequence 5 arrive sequence 6 nucleotide sequence, specific probe have sequence table in sequence 23 nucleosides Acid sequence；There is the specific primer of the trimethoprim class Drug-resistant gene dfrB6 sequence 7 in sequence table to arrive sequence The nucleotide sequence of row 8, specific probe have the nucleotide sequence of sequence 24 in sequence table；The trimethoprim class The specific primer of Drug-resistant gene dfrC arrives the nucleotide sequence of sequence 10, specific probe with sequence 9 in sequence table Nucleotide sequence with sequence in sequence table 25；The specificity of the trimethoprim class Drug-resistant gene dfrD is drawn Object arrives the nucleotide sequence of sequence 12 with sequence 11 in sequence table, and specific probe has the nucleotide of sequence 26 in sequence table Sequence；There is the specific primer of the trimethoprim class Drug-resistant gene dfrE sequence 13 in sequence table to arrive sequence 14 nucleotide sequence, specific probe have the nucleotide sequence of sequence 27 in sequence table；The trimethoprim class The specific primer of Drug-resistant gene dfrF arrives the nucleotide sequence of sequence 16, specific probe with sequence 15 in sequence table Nucleotide sequence with sequence in sequence table 28；The specificity of the trimethoprim class Drug-resistant gene dfrG is drawn Object arrives the nucleotide sequence of sequence 18 with sequence 17 in sequence table, and specific probe has the nucleotide of sequence 29 in sequence table Sequence；There is the specific primer of the trimethoprim class Drug-resistant gene dfrK sequence 19 in sequence table to arrive sequence 20 nucleotide sequence, specific probe have the nucleotide sequence of sequence 30 in sequence table.
In the present invention：
Multiplexed PCR amplification refers to that PCR amplification system includes 10 kinds of trimethoprim class Drug-resistant bases to be detected The amplimer of cause.All primers in system can carry out expanding with the template in sample under same amplification condition anti- It answers.
Asymmetric PCR amplification refers to that concentration ratio by adjusting forward primer and reverse primer and Tm value differences are different, is made The amount of forward primer is less than reverse primer, and improves annealing temperature in the second stage of amplification so that forward primer is moved back at this At fiery temperature can not with template renaturation, and reverse primer due to Tm values be higher than sense primer, still can be normal under the annealing temperature With template renaturation and further extend, the final asymmetric expanding effect for realizing that the yield of two chains in pcr amplification product is different, The single-stranded quantity that can wherein hybridize with trimethoprim class Drug-resistant gene-specific probe is higher than the complementary strand of the chain.
The hybridization reaction includes the following steps：
1) pcr amplification product is mixed with hybridization solution, high-temperature denatured and quickly cooling is so that double stranded PCR products unwinding is single-stranded；
2) hybridization mixture after above-mentioned denaturation hybridizes a timing with the probe being fixed on carrier at a suitable temperature Between；
3) it is cleaned in washing lotion, removes the hybridization solutions ingredients such as PCR product, the salt ion not hybridized with the probe on carrier.
The hybridization solution includes salting liquid, the surfactant needed for hybridization, and for controlling hybridization rigor Formamide.
The method and its kit of detection trimethoprim class Drug-resistant gene of the present invention, have integrated Degree height, high sensitivity, specificity are good, and testing result is reliable and stable, and feature at low cost can be widely used for trimethoprim Distribution, the epidemiological surveillance of class Drug-resistant gene are the rational use of medicines, effectively reduce and drug resistant generation is delayed to provide effectively Tool.
Description of the drawings
The specific implementation mode of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is that the probe specificity of 10 kinds of trimethoprim class Drug-resistant genes screens dot matrix layout viewing；
Fig. 2 is the hybridization reaction result figure of the probe specificity screening of 10 kinds of trimethoprim class Drug-resistant genes；
Fig. 3 is the probe dot matrix layout viewing for detecting trimethoprim class Drug-resistant gene in clinical sample；
Fig. 4 is the hybridization reaction result figure for detecting trimethoprim class Drug-resistant gene in clinical sample.
Specific implementation mode
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：
Embodiment 1 detects trimethoprim class Drug-resistant gene using the present invention
The present embodiment has detected all trimethoprim class Drug-resistant genes according to the present invention.
1, the preparation of Oligonucleolide primers and probe
The reference sequences that the present invention is provided according to CARD databases, in conjunction with the sequence retrieved in Genbank databases, needle 3-5 is devised to specificity amplification primer to each involved trimethoprim class Drug-resistant gene, then passes through reality It tests and therefrom filters out best specific primer sets (the sequence 1- sequences 20 as shown in appended sequence table)；According to involved every It is different to devise 1-5 items for each drug resistant gene for a kind of sequence-specific of trimethoprim class Drug-resistant gene Specific probe, then sensitivity and specificity are therefrom filtered out by experiment and are satisfied by the specific probe of requirement (as appended The sequence of sequence 21- shown in sequence table 30).
Reverse primer used in the present embodiment be design specific reverse primers sequence (i.e. sequence 2,4,6,8, 10,12,14,16,18,20) 5 '-ends add the preceding paragraph and cls gene to be checked not no unrelated sequences of homology completely, and mark Remember fluorescent molecular：As 5 '-TAMRA-cgcggcgtgctgtggtgtttggtg- specific primer sequences.So that reverse primer exists It can still continue to expand under the higher anneal temperature of asymmetric PCR second stage amplification, and the single stranded end amplified is taken With fluorescent molecular, after the single stranded product and specific probe hybridization so that specific probe position shows fluorescence.
Specific probe used in the present embodiment is the 5 '-of the specific probe sequence (i.e. sequence 21-30) designed End connects upper 25 poly (T), and is chemically modified in end：As NH2-poly(T)25Specific probe sequence.Make Specific probe can be fixed on by chemical combination key on carrier, and reduce influence of the steric hindrance to hybridization.
Detection trimethoprim class Drug-resistant gene-specific primer used in the present embodiment and probe, by Shanghai Ying Jun Bioisystech Co., Ltd synthesizes.
2, the preparation of trimethoprim class Drug-resistant gene detecting chip
Using GeneMachine point sample instruments, oligonucleotide probe (that is, specific probe) is dissolved in 50% (V/ respectively V) (concentration and probe concentration is 10 μM) in DMSO solution.Probe dot matrix arrangement mode as shown in Figure 1 exists dissolved probe points system Aldehyde group modified substrate (Optical grade aldehyde radical substrate, CapitalBio Corporation, production number： 420022) on (every dosage is about 0.44nl), after drying and fixing, 12 sample fence (SmartGrid are stickedTM, the rich Austria in Beijing Biochip Co., Ltd, production number：430033) spare.
Note：HybPCP3 shown in Fig. 1 is hybridization positive control probe, and sequence is：5’-gcaaccaccaccggagg.
3. prepared by measuring samples
Laboratory where inventor is for many years from the plasmid for being clinically separated TMP antibody-resistant bacterium and other researchers give It is middle to obtain all 10 kinds of trimethoprims class Drug-resistant gene clonings according to the present invention respectively, to all clones into Row Sanger sequencings, the dfr drug resistant gene reference sequences for determining the dfr drug resistant genes sequence cloned and being provided in CARD databases Unanimously.
Measuring samples are numbered and corresponding dfr gene types are shown in Table 2.
2 measuring samples of table are numbered and corresponding dfr gene types
4.PCR expands dfr genetic fragments
The present embodiment is expanded using cloned plasmids DNA as template using asymmetric PCR.
Asymmetry amplification is carried out to 10 kinds of cloned plasmids respectively with the combination of dfr gene primers, while using being marked on primer Fluorescent molecular TAMRA to PCR product carry out fluorescent marker.
The reaction system of asymmetric PCR amplification is following (to use Tiangeng Products 2 × Taq PCR MasterMix, product Number：KT201), system amplification is carried out according to sample size：
Note：Cloned plasmids DNA refers to one kind in table 2.
PCR reactions carry out on DNA Engine Tetrad 2 (BIO-RAD) thermal cycler, using following thermal cycle journey Sequence：
5. chip hybridization and signal detection
Take amplification after PCR product, with the salting liquid of debita spissitudo, surfactant, formamide and hybridize the positive it is right According to the target mixing of probe, it is configured to and fixed probe is reacted on chip hybridization reaction solution.Single part of hybridization reaction solution Be formulated as follows and (system amplification carried out according to sample size)：
Remarks explanation：Hybridizing positive control sequence is：TAMRA-cctccggtggtggttgc.
The trimethoprim class Drug-resistant gene detecting chip and cover plate (SmartCover that will be preparedTM, Beijing is won Biochip Co., Ltd difficult to understand, production number：430044) it is positioned in hybridizing box.20 μ l hybridization reaction solutions are taken, are sealed after sample-adding Hybridizing box has been closed, 32 DEG C has been placed in and hybridizes 2 hours.After hybridization, chip is taken out, washing lotion (2 × SSC and 0.2%SDS) is placed in In, room temperature, which is shaken, washes 5 minutes；Chip deionized water is rinsed after five minutes later, centrifuge dripping.
Fluorescence signal on chip uses GenePix 4200AL laser confocal scanners and its software kit GenePix Pro.6.0 (Axon Inc.) scanning collection.Sweep parameter is as follows：Excitation wavelength：532nm；Laser Power：33%； PMT：400.
6. interpretation of result
The hybridization signal that each probe signals point is extracted with 6.0 softwares of GenePix Pro, calculates each probe signals The signal-to-noise ratio (Signal Noise Ratio, SNR=(signal value-background value)/background value) of point, so that it is determined that methoxy in sample Benzylamine miazines Drug-resistant gene type prompts drug resistance.When signal-to-noise ratio is more than or equal to 3, judge corresponding to the probe Genetic test result is the positive, and such as all specific probe signal-to-noise ratio are respectively less than 3, then judges not including in the sample to be tested to be measured 10 kinds of trimethoprim class Drug-resistant genes.
The chip hybridization reaction result of 10 kinds of trimethoprim class Drug-resistant genes is as shown in Figure 2.
The results show that in the different specific probe of the 1-5 items of each drug resistant gene design, probe dfrB1-2 (sequence 21) and and only positive signal is generated with the sample TR-21 containing dfrB1 genes；Probe dfrB2-5 (sequence 22) and spy Needle dfrB2-1 with and positive signal, and its middle probe dfrB2-5 (sequences are only generated with the sample TR-22 containing dfrB2 genes 22) sensitivity is more preferable；Probe dfrB3-2 (sequence 23) with and positive letter is only generated with the sample TR-23 containing dfrB3 genes Number；Probe dfrB6-2 (sequence 24) with and only generate positive signal with the sample TR-24 containing dfrB6 genes；Probe dfrC-1 (sequence 25) and and only positive signal is generated with the sample TR-25 containing dfrC genes；Probe dfrD-1 (sequence 26) with and only Positive signal is generated with the sample TR-26 containing dfrD genes；Probe dfrE-2 (sequence 27) with and only with contain dfrE genes Sample TR-27 generate positive signal；Probe dfrF-1 (sequence 28) with and only generated with the sample TR-28 containing dfrF genes Positive signal；Probe dfrG-1 (sequence 29) with and only generate positive signal with the sample TR-29 containing dfrG genes；Probe DfrK-1 (sequence 30) with and only generate positive signal with the sample TR-30 containing dfrK genes.
The chip test result of all samples with it is completely the same listed by table 2, and specific probe (sequence 21-30) with and only Positive signal is generated with the sample comprising gene corresponding to probe, and without non-spy between other gene PCR amplified productions Specific hybridization shows high probe specificity.Result and sequencing result obtained by this method is completely the same.Illustrate the present invention The method of offer can specificity detect above-mentioned 10 kinds of trimethoprims class Drug-resistant gene.
Embodiment 2：Trimethoprim class Drug-resistant gene in clinical sample is detected using the present invention
This example demonstrates that method provided by the invention and kit can be used for methoxy to be checked in clinically isolated bacterium bacterial strain The detection of benzylamine miazines Drug-resistant gene.
1. the preparation of Oligonucleolide primers and probe
The Oligonucleolide primers that the present embodiment uses are the same as embodiment 1；The specific probe used is on the basis of embodiment 1 On the specific probe (sequence 21-30) that preferably goes out, method and step are the same as embodiment 1.
2. the preparation of trimethoprim class Drug-resistant gene detecting chip
As shown in Figure 3, other methods and step are the same as embodiment 1 for the probe dot matrix arrangement mode that the present embodiment uses.
3. prepared by measuring samples
The present embodiment to be clinically separated and collect pseudomonas aeruginosa (sample number Pae117), Klebsiella Pneumoniae (sample Article Number Kpn063), staphylococcus aureus (sample number Sau037, Sau126), enterococcus (sample number ENT145), Escherichia coli Samples such as (sample number Eco012) are detected.Through sequencing, sample P ae117 includes dfrB1 genes, and sample Kpn063 includes DfrB3 genes, sample Sau037 include dfrC genes, and sample Sau126 includes dfrG genes, and sample ENT145 includes dfrE bases Cause, sample Eco012 do not include any 10 kinds of trimethoprim class Drug-resistant genes to be detected, are negative control.
To above-mentioned Clinical isolation, using Qiagene companies DNeasy Blood＆Tissue Kit (production numbers： 69506) bacterium full-length genome nucleic acid is extracted.
4.PCR expands dfr genetic fragments
Method and step are the same as embodiment 1.Only the template in PCR amplification system is replaced with by cloned plasmids DNA from above-mentioned The bacterium full-length genome nucleic acid (a concentration of 200ng/ul or so) extracted in Clinical isolation.
5. chip hybridization and signal detection
Method and step are the same as embodiment 1.
6. interpretation of result
The hybridization signal value and background value of each probe signals point are extracted with 6.0 softwares of GenePix Pro, are calculated each The signal-to-noise ratio of a probe signals point, so that it is determined that trimethoprim class Drug-resistant gene to be detected in sample there are feelings Condition is prompted to trimethoprim class drug resistance.
The chip hybridization reaction result of measuring samples is as shown in Figure 4.The signal-to-noise ratio computation result such as table of above-mentioned measuring samples 3。
Testing result (signal-to-noise ratio) of the table 3. to Clinical isolation
Data analysis shows that only dfrB1 specific probes (sequence 21) results of hybridization is the positive for sample P ae117, Show in sample P ae117 to include dfrB1 drug resistant genes；For sample Kpn063, only dfrB3 specific probes (sequence 23) are miscellaneous Knot fruit is the positive, includes dfrB3 drug resistant genes in display sample Kpn063；For sample Sau037, only dfrC specificity is visited Needle (sequence 25) results of hybridization is the positive, includes dfrC drug resistant genes in display sample Sau037；For sample ENT145, only DfrE specific probes (sequence 27) results of hybridization is the positive, includes dfrE drug resistant genes in display sample ENT145；For sample Product Sau126, only dfrG specific probes (sequence 29) results of hybridization are the positive, include dfrG drug resistances in display sample Sau126 Gene；For sample Eco012, no specific probe hybridization result is the positive, is not included in display sample Eco012 any to be measured 10 kinds of trimethoprim class Drug-resistant genes.
Comparable chip testing result and sequencing result, it can be found that the testing result of two methods is completely the same.Illustrate this The detection of the method 10 kinds of trimethoprim class Drug-resistant genes suitable for Clinical isolation provided is provided.
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair Bright to be not limited to above example, acceptable there are many deformations.Those skilled in the art can be from present disclosure All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Sequence table
<110>Zhejiang University
<120>Detect ten kinds of trimethoprim class Drug-resistant genetic methods and used kit
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tgtattccca tcgaacgcca 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
caagcgccgc aacaggataa 20
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
atgaagccaa cgctcccg 18
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
cttgaccctg ccaagcgg 18
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
agttgctggc cagtttgcg 19
<210> 6
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
ccacgcgttc aagcgca 17
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
taatccagtc gctggccagt 20
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
caagcgcggc aacaggata 19
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
cgctcacgat aaacaaagag 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
tcgaataacg tttgtcctcc 20
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
gcaaggataa cgacattcca t 21
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
tatatctgtt ctcccccgaa 20
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
agaagatgtg ccttcaatgg 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
tttcatgcgc aatcagatgt 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
gtttgattgt tgcgaggtca 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
tacatctcgt cctttagcca 20
<210> 17
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
tttgattgct gcgatggata a 21
<210> 18
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
aggtaaaccc cttatctctc g 21
<210> 19
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
tgcgatggat aagaataggg t 21
<210> 20
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
taacagatac ttcgctccac t 21
<210> 21
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
cgagtctgag gctcacccag 20
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
tcccctgagt gccacctttg 20
<210> 23
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
gtcgagtccg agtctcaccc 20
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
cgcctggcaa ggtcagattg 20
<210> 25
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
gcttcatttc accatgaagg ggt 23
<210> 26
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
tgcccattca atagaagctg ca 22
<210> 27
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
actttatcag cgcagtgccg 20
<210> 28
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
tgttgtttcc accacaacag agt 23
<210> 29
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
ggaggattcc caaggactgg g 21
<210> 30
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 30
tggagaatcc caaaggattg gga 23

Claims (5)

1. the kit of 10 kinds of trimethoprim class Drug-resistant genes is detected, it is characterized in that：
Include the amplimer combination that 10 kinds of trimethoprim class Drug-resistant genes are carried out with specific amplification, sequence is such as SEQ ID NO:Shown in 1-20；
Further include the specific probe for detecting 10 kinds of different trimethoprim class Drug-resistant genes, sequence such as SEQ ID NO:Shown in 21-30.
2. the kit of 10 kinds of trimethoprim class Drug-resistant genes of detection according to claim 1, it is characterized in that： Specific probe is fixed on carrier；The carrier is following any：
Silicon chip, the film derived from various functions group, with the slide carrier of various functions base group modification.
3. the kit of 10 kinds of trimethoprim class Drug-resistant genes of detection according to claim 1 or 2, feature It is：The kit further includes pcr amplification reaction liquid and hybridization reaction solution.
4. the 10 kinds of trimethoprim class Drug-resistants of detection carried out using the kit as described in claims 1 to 3 is any The method of gene, it is characterized in that including the following steps：
1) the amplimer combination for, including using kit and pcr amplification reaction liquid include to that may be present in sample to be tested Asymmetric amplification is carried out at least one of 10 kinds of trimethoprim class Drug-resistant genes to be measured, while PCR is produced Object carries out fluorescent marker；
2), the amplified production obtained by step 1) is mixed with the hybridization reaction solution in kit, then miscellaneous with specific probe progress It hands over；Scanned signal value, the background value for obtaining each specific probe；Calculate the signal-to-noise ratio of every specific probe：Signal-to-noise ratio =(signal value-background value)/background value；When signal-to-noise ratio is more than or equal to 3, judge that the genetic test result corresponding to the probe is The positive, such as all specific probe signal-to-noise ratio are respectively less than 3, then judge not including 10 kinds of emilium tosylates to be measured in the sample to be tested Miazines Drug-resistant gene.
5. the method for 10 kinds of trimethoprim class Drug-resistant genes of detection according to claim 4, it is characterized in that：It waits for Survey 10 kinds of trimethoprim class Drug-resistant genes be：dfrB1、dfrB2、dfrB3、dfrB6、dfrC、dfrD、dfrE、 DfrF, dfrG and dfrK.
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CN110079619A (en) * 2019-04-27 2019-08-02 浙江大学 Detect the specific primer of four kinds of glycopeptide class Drug-resistant genes and probe combinations and application in gram-positive bacterium
CN112649600A (en) * 2021-01-13 2021-04-13 青岛农业大学 DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110079619A (en) * 2019-04-27 2019-08-02 浙江大学 Detect the specific primer of four kinds of glycopeptide class Drug-resistant genes and probe combinations and application in gram-positive bacterium
CN112649600A (en) * 2021-01-13 2021-04-13 青岛农业大学 DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs

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