CN110804667A - Fluorescent quantitative PCR (polymerase chain reaction) complete reagent for detecting tetracycline antibiotic resistance gene and application thereof - Google Patents
Fluorescent quantitative PCR (polymerase chain reaction) complete reagent for detecting tetracycline antibiotic resistance gene and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR reagent set for detecting tetracycline antibiotic drug resistance genes and application thereof. The invention firstly discloses a reagent set for detecting tetracycline antibiotic drug resistance genes, which consists of a reagent I and a reagent II. The invention further discloses a method for detecting whether a sample to be detected contains a tetracycline antibiotic drug-resistant gene. The TaqMan fluorescence quantitative PCR method established by the invention aiming at the tetracycline antibiotic drug resistance gene tetX3 and/or tetX4 has the technical advantages of rapidness, high sensitivity and strong specificity, is suitable for culturable bacteria and non-culturable bacteria, has the same PCR reaction condition, ensures that 2 tetracycline antibiotic drug resistance genes in a sample to be detected can be simultaneously detected under the same PCR condition, and has important significance for monitoring the tetracycline antibiotic drug resistance in the environment.
Description
Technical Field
The invention relates to a molecular biology detection method of bacteria in the technical field of biology, in particular to a fluorescent quantitative PCR reagent set for detecting tetracycline antibiotic drug resistance genes and application thereof.
Background
Tigecycline is a novel broad-spectrum tetracycline antibiotic, is clinically used for treating severe infection caused by multiple drug-resistant bacteria and pan-drug-resistant bacteria, is the last defense line used by the current antibiotics, and is known as 'super antibiotic'. The guidelines published by the ministry of health previously indicate that tigecycline is resistant to superbacteria. Recently, new transferable tigecycline high-level resistance genes tetX3 and tetX4 were first reported in china. The discovery of tigecycline drug resistance genes and their transferable properties pose a serious threat to public health safety and animal husbandry development.
Compared with the common PCR, the Taqman probe method has the advantages of rapidness, convenience and the like. In addition, compared with the dye method, the Taqman probe method avoids the influence of nonspecific amplification and primer dimer, and the specificity and the accuracy are far better than those of the dye method. At larger sample sizes, the cost is even lower than with the dye method. At present, no Taqman probe fluorescent quantitative PCR detection method aiming at drug resistance genes tetX3 (GenBank: MK134375.1) and tetX4 (GenBank: MK134376.1) exists.
Disclosure of Invention
The technical problem to be solved by the invention is how to conveniently and rapidly detect the tetracycline antibiotic drug-resistant genes tetX3 and/or tetX 4.
In order to solve the technical problems, the invention firstly provides a kit for detecting or assisting in detecting tetracycline antibiotic resistance genes.
The kit A is used for detecting or assisting in detecting tetracycline antibiotic resistance genes, the tetracycline antibiotic resistance genes are tetX3 and/or tetX4, and the kit is composed of a reagent I for detecting tetX3 and a reagent II for detecting tetX 4;
the reagent I comprises an upstream primer F1 and a downstream primer R1;
the upstream primer F1 is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown as SEQ ID No. 1;
(a2) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.1 and has the same function as SEQ ID No. 1;
the downstream primer R1 is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown as SEQ ID No. 2;
(a4) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.2 and has the same function with SEQ ID No. 2;
the reagent II comprises an upstream primer F2 and a downstream primer R2;
the upstream primer F2 is (b1) or (b 2):
(b1) a single-stranded DNA molecule represented by SEQ ID No. 4;
(b2) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.4 and has the same function as SEQ ID No. 4;
the downstream primer R2 is (b3) or (b 4):
(b3) a single-stranded DNA molecule represented by SEQ ID No. 5;
(b4) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.5 and has the same function as SEQ ID No. 5.
In the kit, the reagent I further comprises a probe P1;
the probe P1 is (a5) or (a6) as follows:
(a5) a single-stranded DNA molecule represented by SEQ ID No. 3;
(a6) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.3 and has the same function as SEQ ID No. 3;
the reagent II also comprises a probe P2;
the probe P2 is (b5) or (b6) as follows:
(b5) a single-stranded DNA molecule represented by SEQ ID No. 6;
(b6) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.6 and has the same function as SEQ ID No. 6.
The invention also provides application of the kit, which is as follows (1) or (2):
(1) detecting whether a to-be-detected or auxiliary detection sample contains a tetracycline antibiotic resistance gene; the tetracycline antibiotic drug-resistant gene is tetX3 and/or tetX4,
(2) preparing a kit for detecting or assisting in detecting whether a sample to be detected contains a tetracycline antibiotic resistance gene; the tetracycline antibiotic drug resistance gene is tetX3 and/or tetX 4.
Kits containing the above kits are also within the scope of the invention; the kit is used for detecting or assisting in detecting whether a sample to be detected contains a tetracycline antibiotic resistance gene; the tetracycline antibiotic drug resistance gene is tetX3 and/or tetX 4.
The preparation method of the kit comprises the step of packaging each primer separately.
In the kit, the proportion of the primers and the probes in the reagent can be determined by a person skilled in the art according to the detection effect.
The invention also provides a method for detecting or assisting in detecting whether a sample to be detected contains a tetracycline antibiotic resistance gene, wherein the tetracycline antibiotic resistance gene is tetX3 and/or tetX4, and the method comprises the following steps: detecting whether the nucleic acid of a sample to be detected contains a target sequence of the reagent I or the reagent II, if the nucleic acid contains the target sequence of the reagent I, the sample to be detected carries or is candidate to carry a drug resistance gene tetX 3; if the nucleic acid contains the target sequence of the reagent II, the sample to be tested carries or is candidate to carry a drug-resistant gene tetX 4.
The target sequence of the reagent I can be shown as SEQ ID No. 7.
The target sequence of the reagent II can be shown as SEQ ID No. 8.
In the above method, the step of detecting or detecting with the aid of a nucleic acid of a sample to be tested whether the nucleic acid contains a target sequence of reagent I or reagent II is: and (2) respectively carrying out PCR amplification by using a reagent I and a reagent II by using nucleic acid of a sample to be detected as a template, wherein if the PCR amplification can obtain a specific amplification product, the sample to be detected contains or is a candidate of a target sequence containing the reagent, and if the PCR amplification can not obtain the specific amplification product, the sample to be detected does not contain or is not a candidate of the target sequence containing the reagent.
The invention also provides a method for detecting whether a sample to be detected contains a tetracycline antibiotic resistance gene, wherein the tetracycline antibiotic resistance gene is tetX3 and/or tetX4, and the method comprises the following steps: and (3) performing fluorescent quantitative PCR amplification by using nucleic acid of a sample to be detected as a template and respectively using a reagent I and a reagent II, and quantifying tetracycline antibiotic drug resistance genes tetX3 and/or tetX4 in the sample to be detected according to the Ct value.
The quantitative method is characterized in that the Ct value is used as the Y value and substituted into the standard curve equation, and the obtained X value is the logarithm value of the copy number of the drug-resistant gene.
the standard curve equation of the tetX3 drug resistance gene is as follows: y ═ 3.570X + 46.402.
the standard curve equation of the tetX4 drug resistance gene is as follows: Y-3.393X + 43.868.
The reaction system for any one of the above PCR amplifications may specifically be: 0.8. mu.l of upstream primer, 0.8. mu.l of downstream primer, 10. mu.l of 2 XTaq PCR MasterMix, 6.4. mu.l of non-nucleic acid water and 2. mu.l of template. The concentration of the forward primer and the concentration of the reverse primer in the system were both 0.4 pmol/. mu.L. PCR amplification reaction procedure: pre-denaturation at 95 deg.C for 5 min; 30 (denaturation 95 ℃ C., 30 s; annealing 60 ℃ C., 30 s; extension 72 ℃ C., 30 s); extension 72 ℃ for 10 min.
The reaction system for any one of the above fluorescent quantitative PCR amplifications may specifically be: 0.4. mu.L of the upstream primer, 0.4. mu.L of the downstream primer, 0.4. mu.L of the probe, 0.4ul of the Passive Perferencedye (50X),
probe qPCRSuperMix10 μ L, template 1 μ L, and no nucleic acid water added to the system to 20 μ L. The concentrations of the forward primer, the reverse primer and the probe in the system were all 0.2 pmol/. mu.L. The PCR amplification reaction procedure is shown in Table 2.
The invention also provides a kit for detecting or assisting in detecting a tetracycline antibiotic resistance gene, wherein the tetracycline antibiotic resistance gene is tetX3 or tetX4, and the kit is (a) or (b) as follows:
(a) the reagent I for detecting tetX3 described above;
(b) reagent II described above for the detection of tetX 4.
The invention also provides application of the kit, which is as follows (1) or (2):
(1) detecting or assisting to detect whether a sample to be detected contains a tetracycline antibiotic resistance gene, wherein the tetracycline antibiotic resistance gene is tetX3 or tetX 4;
(2) preparing a kit for detecting or assisting in detecting whether a sample to be detected contains a tetracycline antibiotic resistance gene, wherein the tetracycline antibiotic resistance gene is tetX3 or tetX 4.
The kit comprising the kit of parts is also within the scope of the invention.
The invention establishes a corresponding TaqMan fluorescent quantitative PCR method aiming at 2 types of tetracycline antibiotic drug-resistant genes (tetX3 and tetX4), the detection method has the technical advantages of rapidness, high sensitivity and strong specificity, is suitable for culturable bacteria and non-culturable bacteria, and has the same PCR reaction conditions, thereby ensuring that 2 types of tetracycline antibiotic drug-resistant genes in a sample to be detected can be simultaneously detected under the same PCR condition, and having important significance for monitoring the tetracycline antibiotic drug resistance in the environment.
Drawings
FIG. 1 is an electrophoresis diagram of the amplified products of the DNA of the strain to be tested by the reagent I and the reagent II respectively; wherein A is the amplification result of the reagent I on the DNA of the strain to be detected, and B is the amplification result of the reagent II on the DNA of the strain to be detected; lanes X, X1, X2, X3 and X4 are the amplification results of the DNA of positive strains carrying drug-resistant genes tetX, tetX1, tetX2, tetX3 and tetX4 in sequence, lane-is a negative control, and lane M is DNAmarker.
FIG. 2 is the amplification curves of the reagent I and the reagent II for the fluorescent quantitative PCR of the DNA of the strain to be detected respectively; wherein, A is the amplification curve of the fluorescence quantitative PCR of the reagent I to the DNA of the strain to be detected, and B is the amplification curve of the fluorescence quantitative PCR of the reagent II to the DNA of the strain to be detected.
FIG. 3 is an amplification curve of fluorescent quantitative PCR detection of 10-fold serial diluted plasmid standards; wherein, A is the amplification curve of the fluorescent quantitative PCR detection of the tetX3 plasmid standard substance, and B is the amplification curve of the fluorescent quantitative PCR detection of the tetX4 plasmid standard substance.
FIG. 4 is a standard curve of fluorescent quantitative PCR detection of 10-fold serial diluted plasmid standards; wherein, A is a standard curve of the tetX3 plasmid standard product (the standard curve equation is that Y is-3.570X +46.402), and B is a standard curve of the tetX4 plasmid standard product (the standard curve equation is that Y is-3.393X + 43.868).
FIG. 5 shows the result of fluorescent quantitative PCR detection of the strain.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 design and preparation of primers and probes
A large number of primers and probes for detecting 2 tetracycline antibacterial drug resistance genes (tetX3 and tetX4) are obtained by carrying out a large number of sequence analyses and alignments. And performing a preliminary experiment on each primer, and comparing the performances such as sensitivity, specificity and the like to finally obtain a primer group for detecting the drug resistance genes of the 2-class tetracycline antibacterial drugs, wherein the primer group is specifically shown in table 1.
TABLE 12 primer and Probe information for tetracycline antibacterial drug resistance genes
Note: the upstream primer F1, the downstream primer R1 and the probe P1 form a reagent I; the upstream primer F2, the downstream primer R2 and the probe P2 constitute a reagent II.
Example 2 construction of plasmid Standard
1. tetX3 plasmid standard: the DNA molecules shown in SEQ ID No.7 (i.e., the target sequences of the upstream primer F1 and the downstream primer R1) were inserted into the EcoR V site of the pDM19-T vector, and the other sequences of the pDM19-T vector were kept unchanged, to obtain the recombinant plasmid pDM19-T-tetX3, i.e., the tetX3 plasmid standard.
2. tetX4 plasmid standard: the DNA molecules shown in SEQ ID No.8 (i.e. the target sequences of the upstream primer F2 and the downstream primer R2) are inserted into the EcoR V site of the pDM19-T vector, and other sequences of the pDM19-T vector are kept unchanged, so that the recombinant plasmid pDM19-T-tetX4, namely a tetX4 plasmid standard product, is obtained.
Example 3 primer verification and specificity experiments
First, plasmid standard substance verification primer
A sample to be detected: example 2 plasmid standards were prepared.
PCR amplification was performed using the plasmid standard as a template and the reagents I and II in example 1, respectively.
The PCR amplification reaction systems are all as follows: 0.4. mu.L of the forward primer, 0.4. mu.L of the reverse primer, 0.4. mu.L of the probe, 0.4ul of the PassionPerferencedye (50X),
probe qPCR Supermix (available from Beijing Jinweizhi Biotech Co., Ltd., AQ401-01) 10. mu.L, template 1. mu.L, and no nucleic acid water was added to the total volume to 20. mu.L.
The concentrations of the forward primer, the reverse primer and the probe in the system were all 0.2 pmol/. mu.L.
The PCR amplification reaction procedures are all as shown in Table 2:
TABLE 2 real-time fluorescent quantitative PCR reaction procedure
Plasmid standards were replaced with non-nucleic acid water as described above, and all other steps were unchanged as negative controls.
The results show that reagent I can achieve specific amplification of the tetX3 plasmid standard, and cannot achieve amplification of the tetX4 plasmid standard and the negative control. Reagent II allowed specific amplification of tetX4 plasmid standard, and amplification of tetX3 plasmid standard and negative control was not achieved.
The results show that the two groups of reagents provided by the invention have high specificity to the target sequences respectively.
Second, PCR verification
The test samples are shown in Table 3, wherein the positive strains DH5 α -tetX, DH5 α -tetX1, DH5 α -tetX2, DH5 α -tetX3 and DH5 α -tetX4 are recombined and constructed by the following method:
1) artificially synthesizing tetracycline antibiotic drug-resistant genes tetX (the nucleotide sequence of which is shown in SEQ ID No. 9), tetX1 (the nucleotide sequence of which is shown in SEQ ID No. 10), tetX2 (the nucleotide sequence of which is shown in SEQ ID No. 11), tetX3 (the nucleotide sequence of which is shown in SEQ ID No. 12) and tetX4 (the nucleotide sequence of which is shown in SEQ ID No. 13) respectively;
2) constructing a recombinant plasmid: inserting tetracycline antibiotic resistance genes tetX, tetX1, tetX2, tetX3 and tetX4 into the Ndel recognition site and the Hindll recognition site of a pET-28a (+) vector to obtain recombinant plasmids pET-28a-tetX, pET-28a-tetX1, pET-28a-tetX2, pET-28a-tetX3 and pET-28a-tetX 4;
3) the recombinant bacteria are constructed by transfecting pET-28a-tetX, pET-28a-tetX1, pET-28a-tetX2, pET-28a-tetX3 and pET-28a-tetX4 into Escherichia coli DH5 α (Beijing Bomadder Biotechnology company) respectively to obtain positive strains DH5 α -tetX, DH5 α -tetX1, DH5 α -tetX2, DH5 α -tetX3 and DH5 α -tetX 4.
TABLE 3 information on the strains to be tested
Genomic DNA of each positive strain was extracted, and PCR amplification was performed using the genomic DNA as a template and the reagents I and II in example 1, respectively.
The PCR amplification reaction systems (20. mu.l) were all: 0.8. mu.l of the upstream primer, 0.8. mu.l of the downstream primer, 10. mu.l of 2 XTAQCRMSTERmix (available from Nanjing Novowed Biotech Co., Ltd., product No. P111-01), 2. mu.l of the template, and 20. mu.l of the nucleic acid-free water were added to the system. The concentration of the forward primer and the concentration of the reverse primer in the system were both 0.4 pmol/. mu.L.
The PCR amplification reaction procedures are as follows: pre-denaturation at 95 deg.C for 5 min; 30 (denaturation 95 ℃ C., 30 s; annealing 60 ℃ C., 30 s; extension 72 ℃ C., 30 s); extension 72 ℃ for 10 min.
The genomic DNA of the positive strain was replaced with the nucleic acid-free water according to the above method, and the negative control was prepared without changing the other steps.
The results are shown in FIG. 1, indicating that: reagent I can achieve specific amplification of tetX3 gene, and cannot achieve amplification of the remaining drug-resistant genes and negative controls (shown in fig. 1 a); reagent II allowed specific amplification of the tetX4 gene, but failed to amplify the remaining drug-resistant genes and negative controls (shown in FIG. 1 as B).
Third, fluorescent quantitative PCR verification
A sample to be detected: see table 3.
Genomic DNA of each positive strain was extracted, and fluorescent quantitative PCR amplification was performed using the genomic DNA as a template and the reagents I to II in example 1, respectively.
The PCR amplification reaction systems are all as follows: 0.4. mu.L of the forward primer, 0.4. mu.L of the reverse primer, 0.4. mu.L of the probe, 0.4ul of the PassionPerferencedye (50X),
probe qPCR Supermix (available from Beijing Jinweizhi Biotech Co., Ltd., AQ401-01) 10. mu.L, template 1. mu.L, and no nucleic acid water was added to the total volume to 20. mu.L. The concentrations of the forward primer, the reverse primer and the probe in the system were all 0.2 pmol/. mu.L.
The PCR amplification reaction procedure is shown in Table 2.
The genomic DNA of the positive strain was replaced with the nucleic acid-free water according to the above method, and the negative control was prepared without changing the other steps.
The results show that reagent I can achieve specific amplification of the tetX3 gene, and cannot achieve amplification of the remaining drug-resistant genes and negative controls (shown in A in FIG. 2); reagent II allowed specific amplification of the tetX4 gene, but failed to amplify the remaining drug-resistant genes and negative controls (shown in FIG. 2 as B).
Example 4 creation of Standard Curve
The 2 plasmid standards prepared in example 2 were used to establish a standard curve, respectively.
1. The copy number of the plasmid standard was calculated as follows:
Copies/μL＝(L×C×10-9)/(N×M)；
l: afuga de Ro constant (6.02x 10)23/mol)；
C: concentration of plasmid DNA (ng/. mu.L);
n: recombinant plasmid length (bp);
m: average molecular weight of each base pair (660/bp).
2. Diluting the plasmid standard substance with diluent EASY Dilution (purchased from Takara Bio-engineering (Dalian) Co., Ltd., product number H9160S) special for real-time fluorescence quantitative PCR standard to different gradient concentrations (10)-1ng/μL、10-2ng/μL、10-3ng/μL、10-4ng/μL、10-5ng/μL、10-6ng/μL、10-7ng/μL、10-8ng/μL、10-9ng/. mu.L and 10-10ng/μL)。
3. Carrying out fluorescent quantitative PCR on tetX3 plasmid standard substances with different concentrations by using a reagent I; fluorescent quantitative PCR was performed on tetX4 plasmid standards at different concentrations using reagent II.
Fluorescent quantitative PCR reaction system: 0.4. mu.L of the forward primer, 0.4. mu.L of the reverse primer, 0.4. mu.L of the probe, 0.4ul of the PassionPerferencedye (50X),
probe qPCR SuperMix (available from Beijing Jinweizhi Biotech Co., Ltd., product No. AQ401-01) 10. mu.L, template 1. mu.L, and no nucleic acid water was added to the system to 20. mu.L. The concentrations of the forward primer, the reverse primer and the probe in the system were all 0.2 pmol/. mu.L.
The fluorescent quantitative PCR reaction procedure is shown in Table 2.
The amplification curve of the obtained plasmid standard fluorescent quantitative PCR detection is shown in FIG. 3: a is the amplification curve of the fluorescent quantitative PCR detection of the tetX3 plasmid standard, and B is the amplification curve of the fluorescent quantitative PCR detection of the tetX4 plasmid standard.
According to the amplification curve, with a Ct value (Threshold Cycle: the number of cycles that the fluorescence signal in each reaction tube passes when reaching a set Threshold) as an ordinate and the logarithm of the initial template copy number as an abscissa, a linear relationship exists between the two, a standard curve is prepared by using the Ct value obtained by PCR reaction and the known logarithm of the initial template copy number, and the standard curve of the fluorescence quantitative PCR detection of the plasmid standard is obtained as shown in FIG. 4: a is the standard curve of the tetX3 plasmid standard product (the standard curve equation is that Y is-3.570X +46.402), and B is the standard curve of the tetX4 plasmid standard product (the standard curve equation is that Y is-3.393X + 43.868). Wherein Y is a Ct value obtained by PCR reaction; x is the logarithm of the copy number of the gene of interest.
According to the method, the Ct value range and the standard curve equation of the target gene can be detected, and the detection ranges of the tetX3 drug-resistant gene and the tetX4 drug-resistant gene are respectively as follows: 1.49X 102～1.49×1010copies/μL、1.23×102～1.23×1010copies/μL。
Example 5 actual testing of samples
Bacterial strain detection
A sample to be detected: see table 4. The strain is publicly available at the university of agriculture in china.
TABLE 4 information on the strains to be tested
Note: "-" indicates that tetX3 and tetX4 were negative and the drug resistance gene was determined by whole genome sequencing.
And extracting the genome DNA of the sample to be detected, and performing fluorescent quantitative PCR on the template by respectively adopting a reagent I and a reagent II by taking the genome DNA as the template, wherein the reaction system and the reaction procedure of the fluorescent quantitative PCR are the same as those in example 4.
If positive amplification can be achieved, the strain is a strain positive to the drug-resistant gene, and if positive amplification cannot be achieved, the strain is a strain negative to the drug-resistant gene.
And substituting the Ct value into the standard curve equation obtained in the embodiment 4 to calculate the abundance of the drug-resistant gene in the sample.
The results of the fluorescent quantitative PCR are shown in Table 5, and the results are consistent with the strain information. The copy numbers of tetX3 and tetX4 in the strains are shown in FIG. 5.
TABLE 5 fluorescent quantitative PCR assay results for tetracycline antibiotic resistance gene tetX3/tetX4
The results show that the primer combination and the established detection method can realize the detection of tetracycline antibiotic drug-resistant genes tetX3 and tetX4 in a sample under the same PCR condition, and the results are reliable.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
SEQUENCE LISTING
<110> university of agriculture in China
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acaaaaaatg taaagcccga aaatcgattt gacaatcctg aaataaacag aaatgactta 360
agggctatct tgttgaatag tttagaaaac gacacggtta tttgggatag aaaacttgtt 420
atgcttgaac ctggtaagaa gaagtggaca ctaacttttg agaataaacc gagtgaaaca 480
gcagatttgg ttattcttgc caatggcggg atgtccaagg taagaaaatt tgttaccgac 540
acggaagttg aagaaacagg tactttcaat atacaagccg atattcatca accagagata 600
aactgtcctg gattttttca gctatgcaat ggaaaccggc taatggcatc tcaccaaggt 660
aatttattat ttgctaaccc caataataat ggtgcattgc attttggaat aagttttaaa 720
acacctgatg aatggaaaaa ccaaacgcag gtagattttc aaaacagaaa tagtgtcgtt 780
gattttcttc tgaaaaaatt ttccgattgg gacgaacgct acaaagaatt gattcatgcg 840
acgttgtcat ttgtaggatt ggctacacgg atatttcctt tagaaaagcc ttggaaaagc 900
aagcgcccat tacccataac aatgattggg gatgccgcac atttgatgcc gccttttgca 960
gggcagggag taaatagtgg gttggtggat gccttgatat tgtctgataa tctagccgat 1020
ggaaaattta atagcattga agaggctgtt aaaaattatg aacagcaaat gtttatctat 1080
ggcaaagaag cacaagaaga atcaactcaa aacgaaattg aaatgtttaa acccgacttt 1140
acgtttcagc aattgttaaa tgtataa 1167
<210>10
<211>1080
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
atggcaaact tgttacaaca aaacggtatt gacattaccg tttacgaaag agatgaaaac 60
ccaaaagcac gagtttgggg cggaacgctt gaccttcaca aaaattcagg acaagaagca 120
atgaaaaaag taggattgtt gcaaacctac tatgatttgg cgctacctat gggcgtaaac 180
tttgctgatg agaagggtaa cattatagca acaagaaacc cgacactcga aaataagttt 240
gacaaccccg aaataaatag aaacgcgttg cgaaaaatgt tgcttggcag cttgaaaaat 300
gacacagttg tttgggatag aaaatctatt gggcttgaac aagaaaacgg aaaatggctg 360
ctacattttg aaaataagcc aactgcattg gccgacttta ttattgtttc caatggtgga 420
atgtctaaaa taagaaattt tgtttcagat aatgaagtcg aagaaacagg tacttttatt 480
attcagggcg acattcctga accagaaacg aactgccctg aattttataa gttgtgcaac 540
aacaatagac taatgaccgc acatcaaggg aatttattag ttgcgaatcc atttaacaac 600
ggaatgttaa cttacggtgt cattttcaaa aagcctgaag aatggaataa tggaaaagga 660
ttagatttta agcccacaaa aagcgtttcc gagttcttga caaacaggtt ttcaaattgg 720
agcaatgaat acaaggagtt aattcgttca acaacttttt tcgttggttt aacaataaaa 780
atatttccgc tagacaaaaa gccttggaag agtaatcgtc cgttacccat aactttaatt 840
ggcgacacag ctcacctaat gccacctttt gcagggcagg gcgtaaacat tggactaatg 900
gacgctttga ttttgtcaga aaatcttaca aacgggaaat ttggaacgat acaaagtgct960
attgatgact atgaacaacg aatgtttgtt tacgcaacag aagcacaagc ggactcgaca 1020
aagaatgaaa tagaaatgcg aaatccgagc tttacttttc aacagctaat gaatgtataa 1080
<210>11
<211>1167
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
atgacaatgc gaatagatac agacaaacaa atgaatttac ttagtgataa gaacgttgca 60
ataattggtg gtggacccgt tggactgact atggcaaaat tattacagca aaacggcata 120
gacgtttcag tttacgaaag agacaacgac cgagaggcaa gaatttttgg tggaaccctt 180
gacctacaca aaggttcagg tcaggaagca atgaaaaaag cgggattgtt acaaacttat 240
tatgacttag ccttaccaat gggtgtaaat attgctgatg aaaaaggcaa tattttatcc 300
acaaaaaatg taaagcccga aaatcgattt gacaatcctg aaataaacag aaatgactta 360
agggctatct tgttgaatag tttagaaaac gacacggtta tttgggatag aaaacttgtt 420
atgcttgaac ctggtaagaa gaagtggaca ctaacttttg agaataaacc gagtgaaaca 480
gcagatttgg ttattcttgc caatggcggg atgtccaagg taagaaaatt tgttaccgac 540
acggaagttg aagaaacagg tactttcaat atacaagccg atattcatca accagagata 600
aactgtcctg gattttttca gctatgcaat ggaaaccggc taatggcatc tcaccaaggt 660
aatttattat ttgctaaccc caataataat ggtgcattgc attttggaat aagttttaaa 720
acacctgatg aatggaaaaa ccaaacgcag gtagattttc aaaacagaaa tagtgtcgtt 780
gattttcttc tgaaagaatt ttccgattgg gacgaacgct acaaagaatt gattcatacg 840
acgttgtcat ttgtaggatt ggctacacgg atatttcctt tagaaaagcc ttggaaaagc 900
aagcgcccat tacccataac aatgattggg gatgccgcac atttgatgcc gccttttgca 960
gggcagggag taaatagtgg gttggtggat gccttgatat tgtctgataa tctagccgat 1020
ggaaaattta atagcattga agaggctgtt aaaaattatg aacagcaaat gtttatctat 1080
ggcaaagaag cacaagaaga atcaactcaa aacgaaattg aaatgtttaa acccgacttt 1140
acgtttcagc aattgttaaa tgtataa 1167
<210>12
<211>1167
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
atgacaatgc gaatagatac agacaaacaa atgaatttac ttagtgataa gaacgttgca 60
ataattggtg gtggacccgt tggactgact atggcaaaat tattacagca aaacggcata 120
gacgtttcag tttacgaaag agacaacgac cgagaggcaa gaatttttgg tggaaccctt 180
gacctacaca aaggttcagg tcaggaagca atgaaaaaag cgggattgtt acaaacttat 240
tatgacttag ccttaccaat gggtgtaaat attgctgatg aaaaaggcaa tattttatcc 300
acaaaaaatg taaagcccga aaatcgattt gacaatcctg aaataaacag aaatgactta 360
agggctatct tgttgaatag tttagaaaac gacacggtta tttgggatag aaaacttgtt 420
atgcttgaac ctggtaagaa gaagtggaca ctaacttttg agaataaacc gagtgaaaca 480
gcagatttgg ttattcttgc caatggtgga atgtcgaaaa taaggagctt tgttaccgac 540
acgcaagttg aagaaaccgg tactttcaac atccaagctg atattcttca accggaaata 600
aactgtcccg gattttttca gctatgcaac ggcaaccgat taatggcggg acatcagggc 660
attttattgt ttgccaatcc caataataat ggtgcattgt atttaggaat tagttttaaa 720
acgcccgatg aatggaaaaa taaaattccc ttagattttc aggacagaaa cagcgttgcc 780
gattttttat tgaaaagatt ttccaaatgg agtgaagttt acaaacaatt aatacgttcg 840
gtatcaacat ttcaatgctt gcccacaagg aaatttcctt tgaacaatga ttggaaaagt 900
aaccgtccat tacccataac aatgattggc gatgctgctc atttgatgtc gccttttgca 960
ggacagggtg taaatacggg attattggat gctttgatat tgtctgaaaa ccttacaaac 1020
ggagaattta caagtattga aaatgccatc gaaaactacg aacaacaaat gtttgtttat 1080
gcaaaagata cgcaggacga atcgacagaa aacgaaaccg aaatgtttag tcccaatttt 1140
tcgtttcaaa aattattgaa tctataa 1167
<210>13
<211>1158
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
atgagcaata aagaaaaaca aatgaattta cttagtgata agaacgttgc aataattggt 60
ggtggacccg ttggactgac tatggcaaaa ttattacagc aaaacggcat agacgtttca 120
gtttacgaaa gagacaacga ccgagaggca agaatttttg gtggaaccct tgacctacac 180
aaaggttcag gtcaggaagc aatgaaaaaa gcgggattgt tacaaactta ttatgactta 240
gccttaccaa tgggtgtaaa tattgctgat gaaaaaggca atattttatc cacaaaaaat 300
gtaaagcccg aaaatcgatt tgacaatcct gaaataaaca gaaatgactt aagggctatc 360
ttgttgaata gtttagaaaa cgacacggtt atttgggata gaaaacttgt tatgcttgaa 420
cctggtaaga agaagtggac actaactttt gagaataaac cgagtgaaac agcagatctg 480
gttattattg ccaatggtgg aatgtctaaa gtaagaaaat ttgttaccga cacggaagtt 540
gaagaaacag gtactttcaa tatacaagcc gatattcatc atccagaggt gaactgtcct 600
ggattttttc agctatgcaa tggaaaccgg ctaatggctg ctcatcaagg taatttatta 660
tttgcgaatc ctaataataa tggtgcattg cattttggaa taagttttaa aacacctgat 720
gaatggaaaa accaaacgca ggtagatttt caaaacagaa atagtgtcgt tgattttctt 780
ctgaaagaat tttccgattg ggacgaacgc tacaaagaac tgattcgtgt gacatcatct 840
tttgtagggt tagcgacacg aatatttccc ttaggtaagt cttggaaaag taagcgtcca 900
ttacccataa cgatgattgg agatgctgct catttgatgc ctccttttgc aggacaaggc 960
gtaaacagcg ggttgatgga tgccttgata ttgtcggata atctgaccaa tgggaaattt 1020
aacagcattg aagaggctat tgaaaattat gaacagcaaa tgtttatcta tggcaaagaa 1080
gcacaagaag aatcaactca aaacgaaatt gaaatgttta aacccgactt tacgtttcag 1140
caattgttaa atgtataa 1158
Claims (10)
1. A complete set of reagents for detecting or assisting in detecting tetracycline antibiotic resistance genes, which is characterized in that: the tetracycline antibiotic resistance gene is tetX3 and/or tetX4, and the kit consists of a reagent I for detecting tetX3 and a reagent II for detecting tetX 4;
the reagent I comprises an upstream primer F1 and a downstream primer R1;
the upstream primer F1 is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown as SEQ ID No. 1;
(a2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.1 and has the same function as SEQ ID No. 1;
the downstream primer R1 is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown as SEQ ID No. 2;
(a4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.2 and has the same function with SEQ ID No. 2;
the reagent II comprises an upstream primer F2 and a downstream primer R2;
the upstream primer F2 is (b1) or (b 2):
(b1) a single-stranded DNA molecule represented by SEQ ID No. 4;
(b2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.4 and has the same function as SEQ ID No. 4;
the downstream primer R2 is (b3) or (b 4):
(b3) a single-stranded DNA molecule represented by SEQ ID No. 5;
(b4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.5 and has the same function as SEQ ID No. 5.
2. The kit of claim 1, wherein:
the reagent I also comprises a probe P1;
the probe P1 is (a5) or (a6) as follows:
(a5) a single-stranded DNA molecule represented by SEQ ID No. 3;
(a6) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.3 and has the same function with SEQ ID No. 3;
the reagent II also comprises a probe P2;
the probe P2 is (b5) or (b6) as follows:
(b5) a single-stranded DNA molecule represented by SEQ ID No. 6;
(b6) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID No.6 and has the same function as SEQ ID No. 6.
3. Use of a kit according to claim 1 or 2, characterized in that: the application is (1) or (2) as follows:
(1) detecting or assisting to detect whether a sample to be detected contains a tetracycline antibiotic resistance gene, wherein the tetracycline antibiotic resistance gene is tetX3 and/or tetX 4;
(2) preparing a kit for detecting or assisting in detecting whether a sample to be detected contains a tetracycline antibiotic resistance gene, wherein the tetracycline antibiotic resistance gene is tetX3 and/or tetX 4.
4. Kit comprising the kit of parts according to claim 1 or 2, characterized in that: the kit is used for detecting or assisting in detecting whether a sample to be detected contains a tetracycline antibiotic resistance gene, wherein the tetracycline antibiotic resistance gene is tetX3 and/or tetX 4.
5. A method for detecting or assisting in detecting whether a sample to be detected contains a tetracycline antibiotic resistance gene is characterized in that: the tetracycline antibiotic resistance gene is tetX3 and/or tetX4, the method comprises the following steps: detecting whether the nucleic acid of a sample to be tested contains the target sequence of the reagent I or the reagent II in the claim 1 or 2, if the nucleic acid contains the target sequence of the reagent I, the sample to be tested carries or is candidate to carry a drug-resistant gene tetX 3; if the nucleic acid contains the target sequence of the reagent II, the sample to be tested carries or is candidate to carry a drug-resistant gene tetX 4.
6. The method of claim 5, wherein: the detection or auxiliary detection of whether the nucleic acid of the sample to be detected contains the target sequence of the reagent I or the reagent II is as follows: and (2) respectively carrying out PCR amplification by using a reagent I and a reagent II by using nucleic acid of a sample to be detected as a template, wherein if the PCR amplification can obtain a specific amplification product, the sample to be detected contains or is a candidate of a target sequence containing the reagent, and if the PCR amplification can not obtain the specific amplification product, the sample to be detected does not contain or is not a candidate of the target sequence containing the reagent.
7. A method for detecting whether a sample to be detected contains a tetracycline antibiotic resistance gene is characterized in that: the tetracycline antibiotic resistance gene is tetX3 and/or tetX4, the method comprises the following steps: and (3) performing fluorescent quantitative PCR amplification by using nucleic acid of a sample to be detected as a template and respectively using a reagent I and a reagent II, and quantifying tetracycline antibiotic drug resistance genes tetX3 and/or tetX4 in the sample to be detected according to the Ct value.
8. A complete set of reagents for detecting or assisting in detecting tetracycline antibiotic resistance genes, which is characterized in that: the tetracycline antibiotic resistance gene is tetX3 or tetX4, and the kit is (a) or (b) as follows:
(a) reagent I for the detection of tetX3 as claimed in claim 1 or 2;
(b) reagent II for the detection of tetX4 as claimed in claim 1 or 2.
9. Use of a kit according to claim 8, characterized in that: the application is (1) or (2) as follows:
(1) detecting or assisting to detect whether a sample to be detected contains a tetracycline antibiotic resistance gene, wherein the tetracycline antibiotic resistance gene is tetX3 or tetX 4;
(2) preparing a kit for detecting or assisting in detecting whether a tetracycline antibiotic resistance gene exists in a sample to be detected, wherein the tetracycline antibiotic resistance gene is tetX3 or tetX 4.
10. A kit comprising the kit of claim 8.
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| CN201911132048.7A CN110804667B (en) | 2019-11-18 | 2019-11-18 | Fluorescent quantitative PCR (polymerase chain reaction) complete reagent for detecting tetracycline antibiotic resistance gene and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877409A (en) * | 2021-02-01 | 2021-06-01 | 常州市疾病预防控制中心 | RPA reagent and device for detecting antibiotic resistance gene and detection method thereof |
| CN116063537A (en) * | 2022-12-09 | 2023-05-05 | 深圳市儿童医院 | Monoclonal antibody capable of binding TET (X2) protein with high affinity, and product and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257509A (en) * | 2019-08-02 | 2019-09-20 | 浙江大学 | Tigecycline drug resistant gene family tetX multiple PCR detection kit |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257509A (en) * | 2019-08-02 | 2019-09-20 | 浙江大学 | Tigecycline drug resistant gene family tetX multiple PCR detection kit |
Non-Patent Citations (1)
| Title |
|---|
| TAO HE等: "Emergence of plasmid-mediated high-level tigecycline resistance genes in animals and humans", 《NATURE MICROBIOLOGY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877409A (en) * | 2021-02-01 | 2021-06-01 | 常州市疾病预防控制中心 | RPA reagent and device for detecting antibiotic resistance gene and detection method thereof |
| CN112877409B (en) * | 2021-02-01 | 2023-04-07 | 常州市疾病预防控制中心 | RPA reagent and device for detecting antibiotic resistance gene and detection method thereof |
| CN116063537A (en) * | 2022-12-09 | 2023-05-05 | 深圳市儿童医院 | Monoclonal antibody capable of binding TET (X2) protein with high affinity, and product and application thereof |
| CN116063537B (en) * | 2022-12-09 | 2023-12-29 | 深圳市儿童医院 | Monoclonal antibody capable of binding TET (X2) protein with high affinity, and product and application thereof |
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