CN104531877A - Bacterial drug resistance screening PCR chip - Google Patents

Bacterial drug resistance screening PCR chip Download PDF

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CN104531877A
CN104531877A CN201410856255.8A CN201410856255A CN104531877A CN 104531877 A CN104531877 A CN 104531877A CN 201410856255 A CN201410856255 A CN 201410856255A CN 104531877 A CN104531877 A CN 104531877A
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oxa
tet
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pcr
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雷向东
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Shanghai elephant Biological Technology Co., Ltd.
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NANJING ANKUO MEDICAL TECHNOLOGY Co Ltd
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a bacterial drug resistance screening PCR chip. The bacterial drug resistance screening PCR chip comprises an integrated PCR carrier and 190 pairs (380 pieces) of amplification primer pair sequences used for amplifying genes relevant to bacterial drug resistance. The bacterial drug resistance screening PCR chip has the advantages that the real-time fluorescence PCR technology is adopted for detecting 190 target spots of 170 drug resistant genes of different types at a time, bacteria isolation and cultivation are not needed, and the bacterial drug resistance genes in composite samples can be screened rapidly, accurately and flexibly only in two hours.

Description

A kind of bacterial drug resistance examination pcr chip
Technical field
The invention belongs to biological technical field, be specifically related to a kind of bacterial drug resistance examination pcr chip.
Background technology
As everyone knows, bacterium easily produces resistance to antibiotic.The mechanism that bacterium produces resistance can be divided into two classes substantially, and one is itself have resistant gene or create resistant mutation, claims natural bacterial drug resistance; Two is that bacterium passes through to merge or transform mode acquisition drug resistance gene, and drug tolerant bacteria also can obtain new drug resistance gene from other drug tolerant bacteria, thus produces Multidrug resistance bacterium.
Bacterial drug resistance genes can be divided into multiclass according to the function of drug resistance gene, enumerates a few class as follows:
(1) coding antibiotic modifying enzyme, as aminoglycoside, acetyltransferases, aminoglycoside, adenylyltransferases, aminoglycoside phosphotransferases, beta-lactamase etc., modify by carrying out acetylize, polyadenylation, phosphorylation etc. to antibiotic antibiotic was lost efficacy;
(2) key protein (as multidrug efflux pumps) of coding antibiotic discharging system, strengthens cell and antibiotic is got rid of extracellular ability, thus reduces antibiotic in intracellular effective concentration, produces bacterial drug resistance;
(3) coding antibiotic target proteins, due to transgenation, target protein loses the binding ability with antibiotic, causes antibiotic to play a role.
The acquisition of bacterial drug resistance genes makes bacterium to drug resistant, thus conventional antimicrobial element effect decline was even lost efficacy.Therefore before use antibiotic, which drug resistance gene the bacterium detected in patient body obtains, and the expression level of these drug resistance genes how, and doctor can be instructed to adopt which kind of antimicrobial drug type and dosage thereof.
The detection of bacterial drug resistance of the prior art, Normal practice is antibiotic drug sensitive experiment, first needs to be separated and culturing bacterium, and then carries out drug sensitive experiment one by one.Drug sensitive experiment can only determine that bacterium is to a certain antibiotic resistance, and cannot judge the resistance which kind of drug resistant gene causes.This check system still needs a few days to several weeks when single resistance, a small amount of sample, and consuming time longer when carrying out batch detection, and very easily makes mistakes.
Along with the development of detection technique, progressively start to adopt gene tester to detect Antibiotics Related Genes, be mainly divided into the method by hybridization direct-detection bacterial drug resistance genes, and target gene is increased the method that (PCR) detect afterwards.In method by hybridization direct-detection bacterial drug resistance genes, scholar is had to do the research detecting 60 several drug resistance genes by DNA chip, but, the testing conditions of DNA chip needs bacterium to carry out to be separated equally, enlarged culturing, nonetheless, its accuracy, sensitivity, specificity all also still can not be mentioned in the same breath with the PCR method increased to target gene.And normal PCR due to the PCR condition of each gene not quite identical, can only rest in the level that detects one by one; The PCR product of a large amount of resistant gene of large scale integration carrier one-time detection is adopted to have no on the market.
Summary of the invention
The object of the present invention is to provide and a kind ofly large scale integration can detect the bacterial drug resistance examination pcr chip of medicine resistance genes of various bacteria, relative to above-mentioned conventional bacteria Resistance detection means, it is advantageous that:
1. adopt real-time fluorescence quantitative PCR, accuracy, sensitivity, specificity are high
2. adopt large scale integration to detect, 190 kinds of target spots of 170 drug resistance genes within 2 hours, can be detected
3. can be used for the resistance examination of complex samples: the drug resistance gene detecting 1% content under the background that can exist at 99% non-drug-resistant bacteria, without the need to carrying out separation and Culture, can for the microorganism selection resistance to fungicide of mixing sample in complex environment directly or after pre-amplification.
4. provide not only drug resistance gene examination, can help to understand fully mechanism of drug resistance simultaneously
5., without the need to microbial culture, without the need to drug sensitive experiment, accelerate detection speed, reduce cost of labor
6. easy to use, only need mixing PCR MasterMix and distribute upper plate, without the need to marking each gene, greatly simplifying experimental implementation.
Pcr chip provided by the invention, its component comprises: the amplimer pair being respectively used to the amplification gene relevant to bacterial resistance, PCR positive control (PPC), for fixing amplimer to the integrated PCR carrier with carrying PCR reaction.
Preferably, integrated PCR carrier can be 384 hole PCR plate or 2 piece of 96 hole PCR plate.
Preferably, the amplimer for the gene relevant to bacterial resistance that increase is respectively the nucleotide sequence shown in SEQNO:1-380 to sequence.
Preferably, gene amplification primer puts in order as Figure of description 1 on 384 orifice plates, can detect 2 samples.
Preferably, gene amplification primer putting in order as Figure of description 2 on 2 piece of 96 orifice plate.
Preferably, above-mentioned primer pair all works under unified PCR condition.Concrete pcr amplification program is as shown in table 1:
Table 1
Cycle number Step Temperature Time
1 Denaturation 95℃ 5min
Sex change 95℃ 15sec
40 Annealing 60℃ 15sec
Extend 72℃ 20sec
Melting curve analysis program is as shown in table 2:
Table 2
Step Temperature Constant temperature time Heat-up rate
95℃ 5sec 4.4℃/sec
Melting curve 65℃ 1min 2.2℃/sec
95℃ continuous 0.11℃/sec
Cooling 40℃ 30sec
The gene relevant to bacterial resistance of the present invention comprises:
Aminoglycoside Transacetylase genes involved: aacA, aacA1, aacA4, aacA7, aacA29b, aacC1, aacC2, aacC3, aacC4, aac (3)-Id;
Aminoglycoside adenosyl transferase genes involved: aadA4, aadA7, aadA9, aadA10, aadA12, aadD;
Aminoglycoside phosphotransferase genes involved: aph, aphA, aphA-3, aphA-6, aphA-7, aph2, aph (29)-Ib, strA;
Beta lactamase genes involved: ctx-m4, ctx-m26, ctx-m27, ctx-m32, ges-3, kpc-3, per-1, per-2, shv-34, blaTEM-1, blaTLA-1, blaTLA-2, imp-2, imp-5, imp-9, imp-13, imp-16, vim-4, vim-7, ampC, cmy-9, cmy-13, blanps-1, blanps-2, oxa-1, oxa-2, oxa-5, oxa-9, oxa-10, oxa-12, oxa-18, oxa-20, oxa-22, oxa-27, oxa-29, oxa-40, oxa-45, oxa-46, oxa-48, oxa-50, oxa-54, oxa-55, oxa-58, oxa-60, oxa-61, oxa-75,
Chloramphenicol acyl transferase genes involved: cat, cat2, catIII, catA, catB2, catB4, catB6, catB7, catB8, catB9, catP;
Paraxin/R.D. 17345 translocator genes involved: cmlA1, cmxA, fexA, floR;
Hydrophobic peptides genes involved: cmlB;
Pentapeptide family protein genes involved: qnrA3, qnrB1, qnrB4, qnr;
RRNA VITAMIN B4 N6-methyltransgerase genes involved: ermA, ermB, ermD, ermF, erm (TR);
Esterase genes involved: ereA2, ereB;
MFS output pump genes involved: mefA, mefE, mel;
Macrolide 29-phosphotransferase genes involved: mph (B), mph (A), mph, mphB, mph (BM);
Lytic enzyme genes involved: vgh (A);
Streptogramine B lactonase genes involved: vgbB;
ADP-ribosyltransferase genes involved: arr2;
Tetracycline transporter genes involved: tet (A), tetA (C), tetA (J), tetBSR, tet (D), tet (G), tet (H), tet (L), tetA (Y), tet (Z), effJ, tet (V), tet (K), tet (30), tet (33), tet (38);
Tsiklomitsin inactivating protein genes involved: tet (37), tet (X);
GTP associates elongation factor genes involved: tetB (P), tet (M), tet (O), tet (S), tet (W);
Rrna protected protein genes involved: tet (36), tetQ, tet (T);
Tetracycline repressible protein related gene: tetR (31);
Tsiklomitsin opposing genes involved: tet (U);
Phosphoribosyl transferase genes involved: tet (34);
Tetrahydrofolate dehydrogenase genes involved: dfrII, dfrV, dfrVI, dfrXII, dfr13, dfr16, dfr17, dfrA19, dfrB2, dfrD, dhfr, dhfR, dhfrI, dhfrVIII, dhfrIX, dhfrXV;
Dihydropteroic acid synthetic enzyme genes involved: sulI, sulII, sulIII;
Small-sized multiple medicines output pump genes involved: qacB, qacD, qacED1, qacF, qacF, qacG, qacG2, qacH;
Multiple medicines output pump genes involved: acrB, acrD, mexB, mexD, mexF, mexI, mexY, orf11.
The drug resistant gene that table 3 lists our detection and the class of antibiotic related to:
Table 3
Contribution of the present invention is: provide a kind of novelty, practical bacterial drug resistance genes screening method, without the need to carrying out separation and Culture to sample, only need 190 target spots that can detect above-mentioned 170 drug resistance genes for 2 hours quickly and accurately, the bacterial drug resistance screening of mixing sample can be directly used in; Adopt the primer and PCR reaction system optimized, make product have high sensitivity, accuracy and specificity.
Experimental procedure and experimental result
Note: the DNA extraction reagent related in following experiments, PCR MasterMix, random primer all adopt commercially available research reagents.Primer provided by the invention and PCR condition, can matched well most commercial reagent through optimizing.
Accuracy
Sample source: all kinds of antibiotic resistance gene fragments adopting synthetic, be divided into 4 groups and join respectively in the non-drug tolerant bacteria genome extracted, drug resistance gene concentration is about 10%.
Group 1: add sequence number: the drug resistance gene fragment of 1-95
Group 2: add sequence number: the drug resistance gene fragment of 96-190
Step 1. DNA of bacteria is extracted
Commercially available DNA extraction kit or general DNA extraction step is adopted to extract the genomic dna of sample.DNA total amount is about 10ug, and cumulative volume is about 40ul.
Step 2.PCR amplification reaction solution is prepared
DNA solution is diluted 5 times, configure pcr amplification reaction liquid (consumptions of 96 orifice plate 1 plates) by table 4, after mixing, position, every hole adds 20 μ l pcr amplification reaction liquid.
Table 4
Reagent component Consumption
cDNA ★ 100μl
qPCR MasterMix(2×) 1000μl
ddH 2O 900μl
Total Volume 2000μl
Step 3.PCR programming is as shown in table 1
Melting curve analysis programming is as shown in table 2
Step 4. interpretation of result
The data analysing method of experimental result adopts the analysis of the Ct value of real-time fluorescence quantitative PCR as shown in table 5:
Table 5
Gene symbol GB acquisition number Ct value Judge Gene symbol GB acquisition number Ct value Judge
Group 1
aac(3)-Id AY458224 26.28 + mefE AE008470 N/A -
aac(6’)-Im AF337947 26.25 + mefE AF274302 N/A -
aacA M86913 27.44 + mel DQ839391 N/A -
aacA1 AB113580 28.69 + mexB L11616 N/A -
aacA29b AY139599 29.64 + mexD NC_003430 N/A -
aacA4 NC_006352 25.39 + mexD U57969 N/A -
aacA7 AF263520 26.98 + mexF X99514 N/A -
aacC1 AY139604 29.04 + mexI AE004837 N/A -
aacC2 S68058 29.72 + mexY AB015853 N/A -
aacC3 X55652 28.35 + mph DQ839391 N/A -
aacC4 X01385 25.43 + mph(A) NC_006385 N/A -
aadA10 U37105 26.22 + mph(B) AM260957 N/A -
aadA12 AY665771 27.40 + mph(BM) AY167161 N/A -
aadA4 AY138986 27.00 + mphB D85892 N/A -
aadA7 AY463797 25.46 + msr(A) X52085 N/A -
aadA9 AJ420072 27.85 + orf11 NC_006385 N/A -
aadD AB037420 25.78 + oxa-1 AY139600 N/A -
acrB M94248 29.19 + oxa-10 AY115475 N/A -
acrD U12598 26.07 + oxa-12 U10251 N/A -
ampC J01611 26.37 + oxa-18 U85514 N/A -
aph AJ851089 27.32 + oxa-2 NC_007502 N/A -
aph(2’)-Ib AF337947 26.67 + oxa-20 AF024602 N/A -
aph2 U00004 27.37 + oxa-22 AF064820 N/A -
aphA NC_006352 27.33 + oxa-27 AF201828 N/A -
aphA-3 V01547 27.92 + oxa-29 AJ400619 N/A -
aphA-6 X07753 27.47 + oxa-40 AF509241 N/A -
aphA-7 M29953 27.32 + oxa-45 AJ519683 N/A -
arr2 AF205943 29.98 + oxa-46 AF317511 N/A -
blanps-1 NC_006388 29.77 + oxa-48 AY236073 N/A -
blanps-2 NC_003430 28.45 + oxa-5 X58272 N/A -
blaTEM-1 AJ851089 27.42 + oxa-50 AY306130 N/A -
blatla-1 AF148067 25.25 + oxa-54 AY500137 N/A -
blatla-2 NC_006385 26.79 + oxa-55 AY343493 N/A -
cat M11587 25.67 + oxa-58 AY665723 N/A -
cat M35190 29.55 + oxa-60 AF525303 N/A -
cat S48276 27.99 + oxa-61 AY587956 N/A -
cat M58515 28.42 + oxa-75 AY859529 N/A -
cat2 AY509004 25.78 + oxa-9 M55547 N/A -
catA AJ851089 27.74 + per-1 Z21957 N/A -
catB2 AY139601 29.58 + per-2 X93314 N/A -
catB4 AF322577 26.44 + qacB AF053771 N/A -
catB6 AJ223604 25.09 + qacD M37888 N/A -
catB7 AF036933 26.92 + qacEΔ1 NC_006385 N/A -
catB8 AF227506 25.14 + qacF NC_007502 N/A -
catB9 AF462019 27.68 + qacF AY139598 N/A -
catIII X07848 27.85 + qacG Y16944 N/A -
catP U15027 29.88 + qacG2 AJ609296 N/A -
cat-TC U75299 27.00 + qacH Y16945 N/A -
cmlA1 NC_006388 29.46 + qnr AB187515 N/A -
cmlB AF034958 26.28 + qnrA3 DQ058661 N/A -
cmxA AF024666 27.56 + qnrB1 DQ351241 N/A -
cmy-13 AY339625 28.96 + qnrB4 DQ303921 N/A -
cmy-9 AB061794 26.28 + shv-34 AY036620 N/A -
ctx-m-26 AY157676 29.19 + strA NC_004840 N/A -
ctx-m-27 AY156923 25.10 + strB NC_004840 N/A -
ctx-m-32 AJ557142 29.78 + sul3 AY316203 N/A -
ctx-m-4 Y14156 25.77 + sull NC_006388 N/A -
dfr13(drxXIII) Z50802 25.42 + sulII AJ851089 N/A -
dfr16 AY259085 26.48 + tet(30) AF090987 N/A -
dfr17 AY139588 29.12 + tet(31) AJ250203 N/A -
dfrA19 AM234698 27.51 + tet(32) AJ295238 N/A -
dfrB2 DQ839391 28.86 + tet(34) AB061440 N/A -
dfrD Z50141 28.92 + tet(36) AJ514254 N/A -
dfrII AY139601 25.33 + tet(37) AF540889 N/A -
dfrV AY139589 28.16 + tet(38) AY825285 N/A -
dfrVI Z86002 27.80 + tet(M) M21136 N/A -
dfrXII AY139605 27.50 + tet(M) M85225 N/A -
dhfR Z74777 26.27 + tet(M) X04388 N/A -
dhfr J03306 27.64 + tet(M) X90939 N/A -
dhfr1 NC_006385 28.58 + tet(O) Y07780 N/A -
dhfrIX X57730 28.67 + tet(S) L09756 N/A -
dhfrVIII U10186 25.37 + tet(T) L42544 N/A -
dhfrXV Z83311 27.27 + tet(U) U01917 N/A -
effJ(tet(35) AF353562 28.02 + tet(X) M37699 N/A -
ereA2 AF512546 28.62 + tet(Z) AF121000 N/A -
ereB X03988 30.00 + tetA NC_004840 N/A -
erm(A) X03216 26.96 + tetA NC_006388 N/A -
erm(TR) AF002716 25.64 + tetA AJ851089 N/A -
ermA X51472 27.64 + tetA L06940 N/A -
ermB M11180 27.06 + tetA(33) AJ420072 N/A -
ermD M29832 29.81 + tetA(39) AY743590 N/A -
ermF M14730 27.42 + tetA(J) AF038993 N/A -
fexA AJ549214 29.23 + tetA(Y) AF070999 N/A -
floR AF118107 28.75 + tetB(P) L20800 N/A -
ges-3 AY494717 26.64 + tetBSR D12567 N/A -
imp-13 AJ550807 29.67 + tetD L06798 N/A -
imp-16 AJ584652 27.79 + tetG AF133139 N/A -
imp-16 AJ584652 26.95 + tetH AJ245947 N/A -
imp-2 AJ243491 26.05 + tetK U38428 N/A -
imp-5 AF290912 28.23 + tetL U17153 N/A -
imp-9 AY033653 26.41 + tetQ L33696 N/A -
kikA AY046276 29.22 + tetV AF030344 N/A -
kpc-3 AF395881 29.67 + tetW AJ222769 N/A -
mecA AB037671 28.87 + veb-1 AF010416 N/A -
mefA AJ715499 25.05 + vgb(B) AF015628 N/A -
PPC 26.53 + PPC 26.89 +
Group 2
aac(3)-Id AY458224 N/A - mefE AE008470 27.00 +
aac(6’)-Im AF337947 N/A - mefE AF274302 25.51 +
aacA M86913 N/A - mel DQ839391 29.41 +
aacA1 AB113580 N/A - mexB L11616 25.34 +
aacA29b AY139599 N/A - mexD NC_003430 29.65 +
aacA4 NC_006352 N/A - mexD U57969 26.08 +
aacA7 AF263520 N/A - mexF X99514 35.20 +/-
aacC1 AY139604 N/A - mexI AE004837 25.64 +
aacC2 S68058 N/A - mexY AB015853 26.43 +
aacC3 X55652 N/A - mph DQ839391 28.68 +
aacC4 X01385 N/A - mph(A) NC_006385 29.47 +
aadA10 U37105 N/A - mph(B) AM260957 27.89 +
aadA12 AY665771 N/A - mph(BM) AF167161 29.64 +
aadA4 AY138986 N/A - mphB D85892 26.92 +
aadA7 AY463797 N/A - msr(A) X52085 28.24 +
aadA9 AJ420072 N/A - orf11 NC_006385 25.35 +
aadD AB037420 N/A - oxa-1 AY139600 27.85 +
acrB M94248 N/A - oxa-10 AY115475 29.11 +
acrD U12598 N/A - oxa-12 U10251 25.22 +
ampC J01611 N/A - oxa-18 U85514 29.06 +
aph AJ851089 N/A - oxa-2 NC_007502 25.15 +
aph(2’)-Ib AF337947 N/A - oxa-20 AF024602 25.25 +
aph2 U00004 N/A - oxa-22 AF064820 25.99 +
aphA NC_006352 N/A - oxa-27 AF201828 27.47 +
aphA-3 V01547 N/A - oxa-29 AJ400619 26.69 +
aphA-6 X07753 N/A - oxa-40 AF509241 26.15 +
aphA-7 M29953 N/A - oxa-45 AJ519683 28.62 +
art2 AF205943 N/A - oxa-46 AF317511 25.83 +
blanps-1 NC_006388 N/A - oxa-48 AY236073 28.40 +
blanps-2 NC_003430 N/A - oxa-5 X58272 29.93 +
blaTEM-1 AJ851089 N/A - oxa-50 AY306130 25.04 +
blatla-1 AF148067 N/A - oxa-54 AY500137 29.00 +
blatla-2 NC_006385 N/A - oxa-55 AY343493 26.34 +
cat M11587 N/A - oxa-58 AY665723 27.22 +
cat M35190 N/A - oxa-60 AF525303 26.77 +
cat S48276 N/A - oxa-61 AY587956 26.62 +
cat M58515 N/A - oxa-75 AY859529 28.61 +
cat2 AY509004 N/A - oxa-9 M55547 25.68 +
catA AJ851089 N/A - per-1 Z21957 27.39 +
catB2 AY139601 N/A - per-2 X93314 27.82 +
catB4 AF322577 N/A - qacB AF053771 28.95 +
catB6 AJ223604 N/A - qacD M37888 29.70 +
catB7 AF036933 N/A - qacEΔ1 NC_006385 27.71 +
catB8 AF227506 N/A - qacF NC_007502 26.68 +
catB9 AF462019 N/A - qacF AY139598 29.87 +
catIII X07848 N/A - qacG Y16944 29.83 +
catP U15027 N/A - qacG2 AJ609296 25.32 +
cat-TC U75299 N/A - qacH Y16945 26.52 +
cmlA1 NC_006388 N/A - qnr AB187515 29.46 +
cmlB AF034958 N/A - qnrA3 DQ058661 28.92 +
cmxA AF024666 N/A - qnrB1 DQ351241 28.68 +
cmy-13 AY339625 N/A - qnrB4 DQ303921 25.55 +
cmy-9 AB061794 N/A - shv-34 AY036620 25.52 +
ctx-m-26 AY157676 N/A - strA NC_004840 29.61 +
ctx-m-27 AY156923 N/A - strB NC_004840 28.36 +
ctx-m-32 AJ557142 N/A - sul3 AY316203 28.19 +
ctx-m-4 Y14156 N/A - sulI NC_006388 29.69 +
dfr13(drxXIII) Z50802 N/A - sulII AJ851089 27.52 +
dfr16 AY259085 N/A - tet(30) AF090987 27.14 +
dfr17 AY139588 N/A - tet(31) AJ250203 25.53 +
dfrA19 AM234698 N/A - tet(32) AJ295238 27.63 +
dfrB2 DQ839391 N/A - tet(34) AB061440 25.51 +
dfrD Z50141 N/A - tet(36) AJ514254 27.13 +
dfrII AY139601 N/A - tet(37) AF540889 28.21 +
dfrV AY139589 N/A - tet(38) AY825285 28.20 +
dfrVI Z86002 N/A - tet(M) M21136 25.22 +
dfrXII AY139605 N/A - tet(M) M85225 25.37 +
dhfR Z74777 N/A - tet(M) X04388 29.11 +
dhfr J03306 N/A - tet(M) X90939 27.56 +
dhfr1 NC_006385 N/A - tet(O) Y07780 25.54 +
dhfrIX X57730 N/A - tet(S) L09756 25.91 +
dhfrVIII U10186 N/A - tet(T) L42544 25.04 +
dhfrXV Z83311 N/A - tet(U) U01917 25.85 +
effJ(tet(35) AF353562 N/A - tet(X) M37699 26.27 +
ereA2 AF512546 N/A - tet(Z) AF121000 26.70 +
ereB X03988 N/A - tetA NC_004840 25.52 +
erm(A) X03216 N/A - tetA NC_006388 28.33 +
erm(TR) AF002716 N/A - tetA AJ851089 25.87 +
ermA X51472 N/A - tetA L06940 28.12 +
ermB M11180 N/A - tetA(33) AJ420072 25.07 +
ermD M29832 N/A - tetA(39) AY743590 29.18 +
ermF M14730 N/A - tetA(J) AF038993 29.37 +
fexA AJ549214 N/A - tetA(Y) AF070999 26.16 +
floR AF118107 N/A - tetB(P) L20800 25.01 +
ges-3 AY494717 N/A - tetBSR D12567 27.85 +
imp-13 AJ550807 N/A - tetD L06798 25.40 +
imp-16 AJ584652 N/A - tetG AF133139 27.98 +
imp-16 AJ584652 N/A - tetH AJ245947 27.08 +
imp-2 AJ243491 N/A - tetK U38428 26.67 +
imp-5 AF290912 N/A - tetL U17153 28.20 +
imp-9 AY033653 N/A - tetQ L33696 29.78 +
kikA AY046276 N/A - tetV AF030344 29.79 +
kpc-3 AF395881 N/A - tetW AJ222769 25.64 +
mecA AB037671 N/A - veb-1 AF010416 25.96 +
mefA AJ715499 N/A - vgb(B) AF015628 29.20 +
PPC 26.53 + PPC 26.05 +
The criterion detected is: Ct value < 35, and detected result is positive, display "+" number; Ct value 35-40, detected result needs checking further, display " +/-" number; Ct value > 40 or display N/A, detected result is negative, display "-" number.
Accuracy judges:
Group 1: positive sample positive rate 100%, negative sample negative rate 100%.
Group 2: positive sample positive rate 98.96%, negative sample negative rate 100%.
Specificity: the melting curve peak figure of positive findings is all unimodal (Figure of description 3,4), and this product specificity is 100%.
Sensitivity
Sample source: each drug resistant gene fragment randomly drawing 25 kinds of synthetic, add non-drug resistant gene group DNA extraction liquid after mixing, solution-based is because of total concn: 5mg/ml, cumulative volume 40ul.According to the ratio of 0.5%, 1%, 5%, drug resistant gene solution is joined in non-drug resistant gene solution respectively.
Experimental procedure
Omit step 1, sample DNA extracts.All the other steps are with identical above
Experimental result
Experimental result (following table only lists the detected result containing positive gene) as shown in table 6
Table 6
Sequence number Gene symbol GB acquisition number 5％ Judge 1％ Judge 0.5％ Judge
1 aacC1 AY139604 31.88 + 32.51 + 33.25 +
2 aadA10 U37105 33.39 + 33.97 + 36.71 +/-
3 aadA12 AY665771 32.46 + 32.70 + 33.40 +
4 aadA4 AY138986 33.46 + 34.43 + 35.49 +/-
5 aph2 U00004 31.63 + 31.90 + 34.45 +
6 aphA NC_006352 29.24 + 29.85 + 31.55 +
7 aphA-3 V01547 33.43 + 33.92 + 36.60 +/-
8 aphA-7 M29953 32.63 + 33.57 + 35.78 +/-
9 arr2 AF205943 31.28 + 31.82 + 33.58 +
10 blanps-1 NC_006388 32.85 + 33.50 + 35.45 +/-
11 blanps-2 NC_003430 31.52 + 32.21 + 32.50 +
12 blatla-1 AF148067 33.64 + 33.76 + 36.48 +/-
13 blatla-2 NC_006385 30.20 + 30.60 + 32.43 +
14 cat M58515 33.87 + 34.63 + N/A -
15 qacF NC_007502 30.00 + 30.98 + 30.32 +
16 qacF AY139598 33.91 + 34.52 + 36.38 +/-
17 qacG Y16944 33.15 + 33.52 + 36.17 +/-
28 qacG2 AJ609296 31.95 + 32.36 + 32.77 +
19 qacH Y16945 33.97 + 34.29 + 35.72 +/-
20 qnr AB187515 31.31 + 31.95 + 33.70 +
21 qnrA3 DQ058661 30.04 + 30.77 + 31.52 +
22 tet(31) AJ250203 31.97 + 32.96 + 35.46 +/-
23 tet(34) AB061440 33.80 + 34.01 + 35.09 +/-
24 tetG AF133139 31.58 + 32.21 + 34.21 +
25 tetW AJ222769 29.84 + 30.04 + 31.01 +
26 PPC 31.92 + 32.10 + 32.16 +
Accuracy 100％ 100％ 100％ 54％
Interpretation of result: total gene copy number 5mmol/ml in sample, during drug resistant gene concentration 1%, sensitivity 100%.
Above-mentioned experimental result shows excellent performance of the present invention, can detect the drug resistance gene of under 99% normal background 1%, have applications well prospect.
Embodiment
Embodiment 1: in sputum, bacterial drug resistance genes detects
Samples sources: clinical sputum sample 10 example of Patients With Respiratory Tract Infection
Total DNA extraction in step 1. sputum.
Step 2. increases in advance
Adopt random primer or 190 kinds of primer mixed solutions, DNA extraction thing is increased in advance, makes sample size reach the requirement of cumulative volume 40ul
Step 3.cDNA mixing qPCR MasterMix, is assigned to 96 orifice plates
By cDNA solution dilution 5 times, according to the form below configuration pcr amplification reaction liquid, after mixing, position, every hole adds 20 μ l pcr amplification reaction liquid, and reaction solution composition is as shown in table 4;
Step 4. arranges PCR program
PCR response procedures arranges as shown in table 1;
Melting curve analysis programming is as shown in table 2;
Step 5. interpretation of result
Detected result is imported the excel software write, provide analytical results by software.
Experimental result is as shown in table 7: (following table only lists positive resistant gene detected result and medication guide)
Table 7
Catalogue number(Cat.No.) Positive resistant gene Unsuitable medication
1 - Nothing
2 oax-10 β-lactam antibitics
3 - Nothing
4 - Nothing
5 ermB Quinolones
6 tet(M) Tetracyclines
7 - Nothing
8 - Nothing
9 - Nothing
10 - Nothing
More than display only describes principal character of the present invention and inventive point.Those skilled in the art should understand, and the present invention is not restricted to the described embodiments.Under the prerequisite not departing from inventive point and protection domain, the present invention also has various change, and these changes and improvements all will fall in the scope of protection of present invention.
Specification sheets annex
A. Figure of description totally 3 pages
Accompanying drawing 1:384 hole bacterial drug resistance examination pcr chip primer permutation table
96 hole bacterial drug resistance examination pcr chip primer permutation tables of accompanying drawing 2:2 block combination
Accompanying drawing 3: the melting curve peak figure organizing 1 positive test symbol
Accompanying drawing 4: the melting curve peak figure organizing 2 positive test symbol
B. sequence table totally 5 pages
Sequence table illustrates: the sequence table automatically generated because of Patentin 3.5 has 106 pages more than, and most of blank and duplicate contents on the space of a whole page, consider practical situation, provide the papery sequence table of following form.Contriver submits the E-serial mass color dish of standard txt form to simultaneously.

Claims (5)

1. a bacterial drug resistance examination pcr chip, comprise PCR carrier and the amplimer pair of the gene relevant to bacterial resistance of being respectively used to increase, the described gene relevant to bacterial resistance comprises:
Aminoglycoside Transacetylase genes involved: aacA, aacA1, aacA4, aacA7, aacA29b, aacC1, aacC2, aacC3, aacC4, aac (3)-Id;
Aminoglycoside adenosyl transferase genes involved: aadA4, aadA7, aadA9, aadA10, aadA12, aadD;
Aminoglycoside phosphotransferase genes involved: aph, aphA, aphA-3, aphA-6, aphA-7, aph2, aph (29)-Ib, strA;
Beta lactamase genes involved: ctx-m4, ctx-m26, ctx-m27, ctx-m32, ges-3, kpc-3, per-1, per-2, shv-34, blaTEM-1, blaTLA-1, blaTLA-2, imp-2, imp-5, imp-9, imp-13, imp-16, vim-4, vim-7, ampC, cmy-9, cmy-13, blanps-1, blanps-2, oxa-1, oxa-2, oxa-5, oxa-9, oxa-10, oxa-12, oxa-18, oxa-20, oxa-22, oxa-27, oxa-29, oxa-40, oxa-45, oxa-46, oxa-48, oxa-50, oxa-54, oxa-55, oxa-58, oxa-60, oxa-61, oxa-75,
Chloramphenicol acyl transferase genes involved: cat, cat2, catIII, catA, catB2, catB4, catB6, catB7, catB8, catB9, catP;
Paraxin/R.D. 17345 translocator genes involved: cmlA1, cmxA, fexA, floR;
Hydrophobic peptides genes involved: cmlB;
Pentapeptide family protein genes involved: qnrA3, qnrB1, qnrB4, qnr;
RRNA VITAMIN B4 N6-methyltransgerase genes involved: ermA, ermB, ermD, ermF, erm (TR);
Esterase genes involved: ereA2, ereB;
MFS output pump genes involved: mefA, mefE, mel;
Macrolide 29-phosphotransferase genes involved: mph (B), mph (A), mph, mphB, mph (BM);
Lytic enzyme genes involved: vgh (A);
Streptogramine B lactonase genes involved: vgbB;
ADP-ribosyltransferase genes involved: arr2;
Tetracycline transporter genes involved: tet (A), tetA (C), tetA (J), tetBSR, tet (D), tet (G), tet (H), tet (L), tetA (Y), tet (Z), effJ, tet (V), tet (K), tet (30), tet (33), tet (38);
Tsiklomitsin inactivating protein genes involved: tet (37), tet (X);
GTP associates elongation factor genes involved: tetB (P), tet (M), tet (O), tet (S), tet (W);
Rrna protected protein genes involved: tet (36), tetQ, tet (T);
Tetracycline repressible protein related gene: tetR (31);
Tsiklomitsin opposing genes involved: tet (U);
Phosphoribosyl transferase genes involved: tet (34);
Tetrahydrofolate dehydrogenase genes involved: dfrII, dfrV, dfrVI, dfrXII, dfr13, dfr16, dfr17, dfrA19, dfrB2, dfrD, dhfr, dhfR, dhfrI, dhfrVIII, dhfrIX, dhfrXV;
Dihydropteroic acid synthetic enzyme genes involved: sulI, sulII, sulIII;
Small-sized multiple medicines output pump genes involved: qacB, qacD, qacED1, qacF, qacF, qacG, qacG2, qacH;
Multiple medicines output pump genes involved: acrB, acrD, mexB, mexD, mexF, mexI, mexY, orfll.
2. bacterial drug resistance examination pcr chip as claimed in claim 1, is characterized in that, the right sequence of shown amplimer is respectively the nucleotide sequence shown in SEQ ID NO:1-380.
3. bacterial drug resistance examination pcr chip as claimed in claim 1, it is characterized in that, described amplimer is to being present in pairs respectively in the hole of 384 hole PCR plate.
4. bacterial drug resistance examination pcr chip as claimed in claim 1, it is characterized in that, described amplimer is to being present in the hole of two piece of 96 hole PCR plate respectively in pairs.
5. bacterial drug resistance examination pcr chip as claimed in claim 1, its spy is being, described primer pair can single or the mode that repeats be present in various types of integrated PCR carrier respectively in pairs, namely one block of integrated PCR carrier can detect single or multiple sample, and concrete sample size can be determined with the capacity of PCR carrier according to the actual requirements.
CN201410856255.8A 2014-12-31 2014-12-31 Bacterial drug resistance screening PCR chip Pending CN104531877A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105238870A (en) * 2015-07-03 2016-01-13 中国水产科学研究院黑龙江水产研究所 Primer and kit for detecting aminoglycoside drug resistance genes of aeromonas hydrophila
CN106544445A (en) * 2017-01-11 2017-03-29 博奥生物集团有限公司 For detecting LAMP primer composition and its application of two kinds of drug resistant genes of aminoglycoside-resistant antibiotic
CN107130044A (en) * 2017-06-16 2017-09-05 苏州乔纳森新材料科技有限公司 A kind of molecular labeling and its application for being used to detect riemerella anatipestifer erythromycin-resistant gene
CN113005211A (en) * 2019-12-20 2021-06-22 中国农业大学 LAMP primer and method for detecting tigecycline high-level drug resistance gene tet (X) and variant thereof
CN116313149A (en) * 2023-03-09 2023-06-23 深圳市检验检疫科学研究院 Method and system for detecting bacterial drug resistance

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1396271A (en) * 2001-07-13 2003-02-12 北京三雄高新技术开发有限责任公司 Detection to drug tolerant gene by gene chip technique
CN103555839A (en) * 2013-11-01 2014-02-05 东南大学 Urinary sediment cell kidney fibrosis detection chip based on real-time fluorescent PCR (photo conductive relay)
CN103820552A (en) * 2014-02-26 2014-05-28 东华大学 Real-time quantification PCR chip used for detecting gene expression of mouse cholesterol metabolism

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1396271A (en) * 2001-07-13 2003-02-12 北京三雄高新技术开发有限责任公司 Detection to drug tolerant gene by gene chip technique
CN103555839A (en) * 2013-11-01 2014-02-05 东南大学 Urinary sediment cell kidney fibrosis detection chip based on real-time fluorescent PCR (photo conductive relay)
CN103820552A (en) * 2014-02-26 2014-05-28 东华大学 Real-time quantification PCR chip used for detecting gene expression of mouse cholesterol metabolism

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAFAEL SZCZEPANOWSKI, BURKHARD LINKE等: "Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics", 《MICROBIOLOGY》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105238870A (en) * 2015-07-03 2016-01-13 中国水产科学研究院黑龙江水产研究所 Primer and kit for detecting aminoglycoside drug resistance genes of aeromonas hydrophila
CN105238870B (en) * 2015-07-03 2019-03-22 中国水产科学研究院黑龙江水产研究所 A kind of primer and kit detecting Aeromonas hydrophila aminoglycoside resistant gene
CN106544445A (en) * 2017-01-11 2017-03-29 博奥生物集团有限公司 For detecting LAMP primer composition and its application of two kinds of drug resistant genes of aminoglycoside-resistant antibiotic
CN107130044A (en) * 2017-06-16 2017-09-05 苏州乔纳森新材料科技有限公司 A kind of molecular labeling and its application for being used to detect riemerella anatipestifer erythromycin-resistant gene
CN113005211A (en) * 2019-12-20 2021-06-22 中国农业大学 LAMP primer and method for detecting tigecycline high-level drug resistance gene tet (X) and variant thereof
CN116313149A (en) * 2023-03-09 2023-06-23 深圳市检验检疫科学研究院 Method and system for detecting bacterial drug resistance
CN116313149B (en) * 2023-03-09 2024-04-02 深圳市检验检疫科学研究院 Method and system for detecting bacterial drug resistance

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