CN116555449A - Kit for simultaneously detecting multiple key drug resistance genes based on RPA-LFD and application thereof - Google Patents

Kit for simultaneously detecting multiple key drug resistance genes based on RPA-LFD and application thereof Download PDF

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CN116555449A
CN116555449A CN202211384851.1A CN202211384851A CN116555449A CN 116555449 A CN116555449 A CN 116555449A CN 202211384851 A CN202211384851 A CN 202211384851A CN 116555449 A CN116555449 A CN 116555449A
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primer
probe
mcr
tet
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蒋晗
方结红
吕晨泽
潘雷明
顾秀英
肖朝耿
刘鹏鹏
陈宗伦
黄光荣
唐玉龙
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China Jiliang University
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Abstract

The invention discloses a kit for simultaneously detecting a plurality of key drug resistance genes based on RPA-LFD and application thereof, wherein the kit comprises a primer and a probe aiming at the risk drug resistance genes, and the nucleotide sequences of an upstream primer RPA-F, a downstream primer RPA-R and the probe of a specific RPA primer are shown as SEQ ID NO: 1-SEQ ID NO: shown as 9; the 5 'end of the probe is marked with a fluorescent group, the 3' end is subjected to closed modification, tetrahydrofuran abasic sites are modified in the probe sequence, and primer concentration, magnesium acetate solution concentration, RPA amplification temperature and amplification time are optimized. The invention also provides a lateral chromatography test strip method (LFD), which can be matched with a TSR-200 test strip reader to perform multiple qualitative and semi-quantitative detection on the RPA amplification product. The kit only needs 20-40 minutes in detection time, has high sensitivity, can detect a sample of 10copies/uL at the lowest in the whole course at 37 ℃, has good specificity, and has important significance for the multiple rapid detection of pathogenic bacteria risk drug-resistant genes.

Description

Kit for simultaneously detecting multiple key drug resistance genes based on RPA-LFD and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a kit for simultaneously detecting multiple key drug-resistant genes based on RPA-LFD and application thereof.
Background
The large number of antibiotics used in medicine and agriculture has led to increasingly serious bacterial resistance problems worldwide. When bacteria develop resistance to three or more antibiotics, multiple Drug Resistance (MDR) occurs, resulting in a more severe situation. Among the antibiotics currently available, carbapenems, colistins and tigecyclins are the "last resort" antibiotics for multi-drug resistant bacterial infections, particularly those caused by enterobacteriaceae. Unfortunately, there are increasing reports of the colistin resistance gene mcr-1 (mainly plasmid transmitted), the carbapenem resistance gene blaNDM-1 and the tigecycline resistance gene tet (X4) in enterobacteriaceae pathogens of different sources (e.g. animals, foods and humans). Furthermore, tet (X4) genes are often also present with blaNDM-1 and/or mcr-1 genes, and these plasmid-borne antibiotic resistance genes can also be transferred between epidemic strains of the Enterobacteriaceae family. Therefore, there is a strong need for a rapid, efficient and accurate detection method, while screening and monitoring the drug resistance genes of the enterobacteriaceae pathogens, to control the spread of drug resistance in edible animals, and even the whole food chain, and related environmental populations.
The most widely used molecular-based multi-drug resistance (MDR) -related gene detection methods are the Polymerase Chain Reaction (PCR), including conventional PCR, real-time quantitative PCR, and digital PCR. However, this method requires a complicated instrument for performing complicated thermal cycle and temperature control, and thus is not widely used in this field. To date, researchers have developed new molecular biology techniques, such as isothermal nucleic acid amplification, that can replace PCR for rapid in situ MDR-related gene assessment. Recombinase Polymerase Amplification (RPA), a common isothermal amplification technique of nucleic acids, was first developed by piebenburg et al in 2006, which employs phage recombinases to form complexes with oligonucleotide primers to facilitate the binding of the oligonucleotide primers to homologous sequences of double-stranded DNA molecules; by binding to the single-stranded DNA binding protein and the single-stranded displacement polymerase, it is possible to complete amplification within 30 minutes at a constant temperature of about 37℃and to achieve a high-specificity DNA amplification at a detectable level in a short time with only a small amount of copies of the target DNA. Although RPA is highly dependent on the size of the amplified nucleic acid, the target sequence and primer design, rapid detection of multiple drug resistance by multiplex RPA amplification is still contemplated. The detection of RPA products is mainly visualized by probe fluorescence, gel electrophoresis or nucleic acid lateral chromatography detection test paper (LFD). The LFD immunoassay method is simple and rapid, and the amplified product is accurately detected, so that the method is more suitable for instant detection. In order to meet the requirement of the quantitative detection on the improved precision, a test strip reader and image software can be used for scanning and evaluating the color signals of the LFD.
There is thus an urgent need to develop an RPA-LFD method that would allow the simultaneous, rapid and accurate identification of the critical drug resistance genes mcr-1, blaNDM-1 and tet (X4) in certain enterobacteriaceae pathogens.
Disclosure of Invention
In order to solve the problems, the invention provides a method for simultaneously detecting a plurality of key drug-resistant genes based on RPA-LFD, which is characterized in that multiple primers and probes for drug-resistant genes mcr-1, blaNDM-1 and tet (X4) are designed and screened, the detection is carried out by a method of combining multiple RPA amplifications with nucleic acid lateral chromatography detection test paper (LFD), the concentration of the primers and the concentration of magnesium acetate solution are simultaneously carried out, the temperature and the time of the RPA amplifications are optimized, the rapid and accurate detection of three drug-resistant genes is realized, the detection time is only required to be 20-40 minutes, the sensitivity is high, and a sample of 10copies/uL can be detected at the lowest, and the specificity is good. Has important significance for the rapid detection of key drug-resistant genes mcr-1, blaNDM-1 and tet (X4), cutting off transmission paths and guaranteeing the physical health of people.
The RPA-LFD method adopted by the invention is used for detecting nucleic acid, the target gene is amplified by a recombinase polymerase amplification system (nfo method), and the chromatographic test strip coated with biotin antibody, cy5 antibody and TAMRA antibody is used for detecting in real time, so that the method has the advantages of higher sensitivity, shorter detection time, no instrument dependence, more convenient use and the like compared with other isothermal amplification detection methods.
In one aspect, the invention provides a primer combination for simultaneously detecting three drug resistance genes based on RPA-LFD, wherein the primer combination comprises a forward primer, a reverse primer and a probe, and the sequences of the primer combination are shown in the following table:
drug resistance gene Forward primer Reverse primer Probe with a probe tip
mcr-1 SEQ ID NO：1 SEQ ID NO：2 SEQ ID NO：3
blaNDM-1 SEQ ID NO：4 SEQ ID NO：5 SEQ ID NO：6
tet(X4) SEQ ID NO：7 SEQ ID NO：8 SEQ ID NO：9
The three drug resistant genes are mcr-1, blaNDM-1 and tet (X4).
The mcr-1 gene, the blaNDM-1 gene and the tet (X4) gene are all key drug-resistant genes of pathogenic bacteria of enterobacteriaceae. The mcr-1 gene is a gene which can mediate pathogenic bacteria of enterobacteriaceae to generate drug resistance to polymyxin drugs and can be horizontally transferred through plasmids; the novel metal-beta-lactamase-1 coded by the blaNDM-1 gene can mediate the bacteria to generate drug resistance to carbapenem antibacterial drugs, and the gene can be transferred among various different types of bacteria; the tet (X4) gene can mediate pathogenic bacteria of the enterobacteriaceae family to generate drug resistance to tigecycline drugs. Drug resistance genes mcr-1, blaNDM-1 and tet (X4) mediate bacteria to generate drug resistance to polymyxin, 8 carbapenems and tigecycline antibiotics, thereby seriously threatening the health of human beings and animals.
The mcr-1 gene has a sequence shown in SEQ ID NO:10, the sequence of the blaNDM-1 gene is shown as SEQ ID NO:11, and the tet (X4) gene has the sequence shown in SEQ ID NO: shown at 12.
Further, the 5' ends of the probes of mcr-1, blaNDM-1 and tet (X4) are respectively modified with different groups capable of distinguishing three drug-resistant genes, wherein the groups are biotin, cy5 or TAMRA; performing closed modification at the 3' end, and modifying a tetrahydrofuran abasic site in the probe sequence; digoxin is labeled at the 5' end of the reverse primer of mcr-1, blaNDM-1, tet (X4).
The recombinant enzyme system of the RPA-LFD method adopted by the invention contains endonuclease (nfo protein), the nfo protein can identify and cleave the intermediate modified tetrahydrofuran abasic site of the probe sequence, the sequence after the tetrahydrofuran abasic site in the probe is sheared off, and the 5' end sequence of the probe and the reverse primer form a new amplification product together. The 5 'end of the probe is modified with biotin, cy5 or TAMRA groups, the 5' end of the reverse primer is modified with Digoxin, so that the 5 'end of the amplified DNA fragment carries biotin, cy5 or TAMRA groups, the 3' end carries Digoxin, the amplified products are detected by a test strip coated with biotin antibody, cy5 antibody and TAMRA antibody after 10-15min, and whether the sample contains mcr-1, blaNDM-1 and tet (X4) drug resistance genes can be judged.
The RPA primer and probe designed for the mcr-1, blaNDM-1 and tet (X4) drug resistance genes can be very various, but can be used for multiplex primer amplification, and simultaneously, the primer and probe set for rapidly and accurately detecting the three drug resistance genes of the mcr-1, blaNDM-1 and tet (X4) can be found through a great deal of research. The primer and the probe set provided by the invention can truly realize high-efficiency, convenient, high-sensitivity and high-specificity detection of three drug-resistant genes mcr-1, blaNDM-1 and tet (X4).
Further, the primer combinations are shown in the following table:
in another aspect, the invention provides a kit for simultaneous detection of three drug-resistance genes, the kit comprising a primer combination as described above.
Further, the primer concentration of mcr-1, blaNDM-1 and tet (X4) is 0.15-0.42. Mu.M, and the probe concentration is 0.06-0.12. Mu.M.
Further, the primer and probe concentrations of the mcr-1 gene are preferably 0.30. Mu.M/0.09. Mu.M, the primer and probe concentrations of the blaNDM-1 gene are preferably 0.42. Mu.M/0.12. Mu.M, and the primer and probe concentrations of the tet (X4) gene are preferably 0.30. Mu.M/0.09. Mu.M, wherein the forward primer and reverse primer concentrations are identical.
The chromatography test strip of the RPA-LFD method is provided with a detection line (T line) and a quality control line (C line), and the higher the T/C value (the ratio of the intensity of the T line to the intensity of the C line) in the detection result is, the more obvious the detected positive result is. The higher the T/C value of the detection result, the higher the sensitivity of the detection method, compared to the same sample containing the drug-resistant gene.
According to the invention, the concentrations of the primer and the probe can directly influence the detection result of the drug-resistant gene, when different primer and probe concentrations are used for RPA amplification, the T/C value of the obtained detection result is obviously different when a chromatographic test strip is used for detection, and when the proper primer and probe concentrations are selected, the T/C value is higher, and the detection sensitivity of the drug-resistant gene is also higher.
Further, the kit also comprises RPA reaction dry powder and a test strip; the test strip is embedded with biotin antibody, cy5 antibody and TAMRA antibody.
Further, the RPA reaction dry powder contains nfo protein.
In some embodiments, the invention directly uses commercial RPA reaction dry powder containing kit (TwitAmp nfo kit), in which there are other proteins besides nfo protein, such as recombinase and polymerase.
Furthermore, digoxin antibody is fixed on the gold label pad of the test strip, three detection lines are arranged, biotin antibody, cy5 antibody and TAMRA antibody are respectively fixed, and the detection lines are respectively used for detecting mcr-1, blaNDM-1 and tet (X4) genes.
In some modes, the test strip comprises a gold-labeled pad, a detection line and a quality control line, wherein a Digoxin gold-labeled antibody is fixed on the gold-labeled pad.
Further, the magnesium acetate solution is also contained, and the concentration of the magnesium acetate solution is 5-16.8 mM.
Further, the concentration of the magnesium acetate solution is preferably 14mM.
The magnesium acetate solution is an activator of the RPA isothermal amplification reaction, and proper concentration of the magnesium acetate solution is selected, so that the sensitivity of detecting mcr-1, blaNDM-1 and tet (X4) genes by the RPA-LFD method is further improved.
In yet another aspect, the present invention provides a reagent for simultaneously detecting three drug-resistant genes, the reagent comprising the primer combination as described above.
In yet another aspect, the present invention provides a method for detecting three drug resistance genes using the kit as described above, comprising the steps of:
(1) Extracting nucleic acid to be detected;
(2) Adding RPA reaction dry powder, nucleic acid to be detected and magnesium acetate solution into a reaction tube;
(3) Isothermal amplification;
(4) And detecting by using a test strip, and judging a detection result.
Further, the step (1) of extracting the nucleic acid to be detected is to extract DNA by a boiling method.
In some modes, enterobacteriaceae bacteria liquid such as escherichia coli, campylobacter and the like to be detected is boiled to prepare bacteria liquid template DNA.
In some ways, the present invention improves the boiling process: adding 30 mu L of Tris buffer into 1 mu L of bacteria liquid to be detected, boiling for 5min, cooling for 2min on ice, centrifuging for 12000 Xg for 2min, and taking supernatant as a DNA template to be detected.
The invention adopts an improved boiling method to extract DNA of enterobacteriaceae strains, and can finish DNA extraction only by 10min, and compared with other common boiling methods (about 30 min) or DNA extraction kits (about 30 min) on the market, the invention obviously shortens DNA extraction time.
Further, the isothermal amplification in the step (3) is carried out at the temperature of 25-43 ℃ for 10-30 min.
Performing RPA amplification at 30-50 ℃ respectively on the extracted nucleic acid sample, detecting an amplification product by using an LFD nucleic acid chromatographic test strip, and determining that the optimal isothermal amplification temperature is 25-43 ℃ and most preferably 37 ℃; the amplification time is preferably 20min.
The invention develops a brand new RPA-LFD method which is used for simultaneously identifying key drug resistance genes mcr-1, blaNDM-1 and tet (X4) in certain enterobacteriaceae pathogenic bacteria, wherein the enterobacteriaceae pathogenic bacteria are separated from different food and animal excrement samples. Meanwhile, the invention adopts a TSR-200 colloidal gold reader (Hangzhou O Cheng Yiqi Co., ltd., hangzhou, china) to assist in evaluating the color signals on the test strips, and uses LFD to carry out accurate quantitative analysis, thereby realizing rapid monitoring of key drug resistance genes in enterobacteriaceae pathogenic bacteria in site and/or clinical samples and providing timely and effective information for risk evaluation and establishment of remedial strategies.
Compared with the prior art, the invention has the beneficial effects that:
1. provides a novel method for simultaneously and rapidly quantitatively detecting three drug-resistant genes mcr-1, blaNDM-1 and tet (X4), and rapidly monitors key drug-resistant genes in pathogenic bacteria of enterobacteriaceae;
2. the detection time is short, only 20-40 minutes is needed, the sensitivity is high, the sample of 10copies/uL can be detected at the lowest, and the specificity is good;
3. the method improves the boiling method for extracting the DNA of the enterobacteriaceae strain, and can finish the DNA extraction only by 10 minutes;
4. the method has important significance in realizing the rapid detection of key drug-resistant genes mcr-1, blaNDM-1 and tet (X4) in pathogenic bacteria of enterobacteriaceae in site and/or clinical samples, cutting off transmission paths and guaranteeing the physical health of people.
Drawings
FIG. 1 is a flow chart and a principle description of the detection of multiple drug resistance genes by RPA-LFD in example 1, wherein (a) DNA extraction of Enterobacteriaceae strains; (b) RPA amplification; (c) LFD chromatography lateral flow reagent strip; (d) quantitative analysis;
FIG. 2 is a standard curve of the RPA-LFD test for three drug resistance genes mcr-1, blaNDM-1 and tet (X4) in example 1;
FIG. 3 is a schematic diagram showing the results of RPA-LFD test at different primer concentrations in example 3;
FIG. 4 is a graph showing the change in T/C value of the RPA-LFD test results at different primer concentrations in example 3;
FIG. 5 is a schematic diagram showing the results of RPA-LFD test at different concentrations of magnesium acetate solution in example 4;
FIG. 6 is a graph showing the change in T/C values of the RPA-LFD test results at different concentrations of magnesium acetate solution in example 4;
FIG. 7 is a schematic diagram showing the results of RPA-LFD test at different RPA isothermal amplification temperatures in example 5;
FIG. 8 is a graph showing the change in T/C values of the RPA-LFD test results at different RPA isothermal amplification temperatures in example 5;
FIG. 9 is a schematic diagram showing the results of RPA-LFD test at different RPA isothermal amplification times in example 6;
FIG. 10 is a graph showing the change in T/C values of the RPA-LFD test results at different RPA isothermal amplification times in example 6;
FIG. 11 is a schematic diagram showing the detection results of recombinant E.coli containing different drug resistance genes by the RPA-LFD method in example 8;
FIG. 12 is a schematic diagram showing the result of sensitivity analysis of the RPA-LFD method of example 9 for detecting multiple drug resistance genes.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto. The specific conditions not noted in the examples were carried out according to the conventional conditions or the conditions suggested by the manufacturer, and the reagents or instruments used, not noted by the manufacturer, were conventional products available commercially.
Example 1 method for detecting multiple drug resistance genes by RPA-LFD provided by the invention
The method for detecting the multi-drug resistance gene by using the RPA-LFD provided by the invention comprises the following steps:
1. DNA extraction
1. Mu.L of a bacterial solution of Enterobacteriaceae to be tested was added to 30. Mu.L of a tris buffer solution of 10mM, boiled in a metal bath for 5min, cooled on ice for 2min, centrifuged at 12000 Xg for 2min, and the supernatant was used as a DNA template to be tested (DNA extraction flow is shown in FIG. 1-a). The standard plasmids containing mcr-1, blaNDM-1 and tet (X4) genes were extracted from the standard strains containing mcr-1, blaNDM-1 and tet (X4) genes, respectively, using a kit method, and the plasmid DNA concentrations were determined.
2. RPA isothermal amplification
2. Mu.L of the DNA template to be tested was taken and subjected to RPA isothermal amplification using a TwistAmp nfo KIT (available from TwistDx, model TANFO02 KIT), and the primer and probe sets for three drug resistance genes mcr-1, blaNDM-1 and tet (X4) were as shown in Table 1:
table 1, primer and probe sets of three drug resistance genes
The 5' ends of the reverse primers for mcr-1, blaNDM-1 and tet (X4) were all labeled with Digoxin. Labeling the 5' -end of the probes mcr-1, blaNDM-1 and tet (X4) with biotin, cy5 and TAMRA, respectively; the 3' end of the probe is blocked by a space C3, and the interior of the probe is modified by abasic sites of a nucleotide analogue tetrahydrofuran. The RPA primers and probes were synthesized by Shanghai JieRui bioengineering Co., ltd (Shanghai, china).
The primer/probe concentration for mcr-1 was 0.30. Mu.M/0.09. Mu.M, the primer/probe concentration for blaNDM-1 was 0.42. Mu.M/0.12. Mu.M, and the primer/probe concentration for tet (X4) was 0.30. Mu.M/0.09. Mu.M, where the forward primer and reverse primer concentrations were identical; the concentration of the magnesium acetate solution was 14mM. The RPA reaction system was 50. Mu.L in total, and the commercial kit recommended reaction system was used prior to optimization: consists of 1 tube of RPA reaction dry powder (reconstituted with 29.5. Mu.L buffer), 2.1. Mu.L forward primer (10. Mu.M), 2.1. Mu.L reverse primer (10. Mu.M), 0.6. Mu.L probe (10. Mu.M), 2.5. Mu.L magnesium acetate solution (280 mM), 2.0. Mu.L DNA sample and sterile deionized water up to 50. Mu.L. The RPA isothermal amplification temperature is 37 ℃, and the isothermal amplification time is 20min.
Triple RPA amplification is carried out by a TwistAmp nfo kit to obtain an amplification product containing three drug resistant genes of mcr-1, blaNDM-1 and tet (X4), and the primers and probes of the three genes are designed to make the mcr-1 gene amplification product marked by Digoxin and biotin, the blaNDM-1 gene amplification product marked by Digoxin and Cy5 and the tet (X4) gene amplification product marked by Digoxin and TAMRA (as shown in figure 1-b).
3. Reagent strip detection
(1) Preparation of LFD reagent strips
The LFD reagent strip is formed by continuously superposing a sample pad, a gold-labeled pad, a nitrocellulose filter membrane (NC membrane), an adsorption pad and a bottom plate; the colloidal gold nanoparticles are synthesized by a sodium citrate tannin reduction method, marked by a monoclonal antibody against Digoxin and sprayed on a gold label pad; three detection lines and a quality control line (shown in figure 1-C) are respectively an mcr-1 detection line (T1 line), a blaNDM-1 detection line (T2 line) and a tet (X4) detection line (T3 line), wherein the mcr-1 detection line is coated with antibiotic monoclonal antibody (0.65 mg/mL), the blaNDM-1 detection line is coated with anti-Cy 5 monoclonal antibody (0.3 mg/mL), the tet (X4) detection line is coated with anti-TAMRA monoclonal antibody (0.3 mg/mL), and the quality control line (C line) is coated with goat anti-mouse polyclonal secondary antibody (pAb, 2.0 mg/mL). The immobilized membrane was dried at 37℃for 12h, cut into strips 2.5mm wide with a knife, assembled into LFD strips, and stored in a vacuum bag at room temperature.
The RPA amplification product was diluted 50-fold (2. Mu.L of amplification product was mixed with 98. Mu.L of buffer containing phosphate buffer and 3% Tween 20) and transferred to LFD strips. The labeled RPA product was moved by capillary action on the LFD test strip and was scanned and the T1-T3 line and C line intensities were calculated over 5min with a TSR-200 colloidal gold reader (hangzhou o Cheng Yiqi limited, hangzhou, china).
4. Analysis of detection results
The T/C value is positively correlated with the concentration of the target nucleic acid to be detected. Taking the logarithm of the copy number of the target drug resistance gene of the DNA template to be detected as an X-axis, preparing standard curves of three drug resistance genes, namely mcr-1, blaNDM-1 and tet (X4), according to the T/C value measured on an LFD test strip by a TSR-200 colloidal gold reader as a y-axis, wherein the result is shown in figure 1-d or figure 2 (an enlarged version of the standard curve in figure 1-d), and the standard curve equation is shown in table 2.
Table 2, standard curves for three drug resistance genes, mcr-1, blaNDM-1 and tet (X4)
Drug resistance gene Standard curve R 2
mcr-1 y＝0.1215x-0.0752 0.9881
blaNDM-1 y＝0.1139x-0.081 0.9745
tet(X4) y＝0.1187x+0.051 0.9807
According to the standard curve shown in Table 2, the T/C value has obvious linear correlation with the copy number of the DNA to be detected, and the specific contents of three drug-resistant genes can be quantitatively calculated after the T/C value is measured by adopting an LFD test strip.
The concentration of the target drug-resistant gene can also be calculated according to the following formula according to the copy number of the target drug-resistant gene.
Gene copy number (copies/. Mu.L) = (X/(a. Times.660)). Times.6.02X10. 23
Wherein X is the standard DNA concentration (g/. Mu.L) measured at a wavelength of 260nm and a is the base pair number (bp) of the standard DNA molecule.
Example 2 screening of multiplex RPA amplification primers
The antibiotic resistance gene sequences of the three drug resistance genes mcr-1, blaNDM-1 and tet (X4) were from the gene library (https:// www.ncbi.nlm.nih.gov/gene /). Logging in a gene library, downloading the DNA sequences of mcr-1, blaNDM-1 and tet (X4) drug-resistant genes, carrying out Cluster W homologous alignment on the DNA sequences by using MEG4 software, analyzing the polymorphism of the nucleic acid sequences, and determining a conserved region. The RPA primers and probes were designed for the conserved region sequences, combined with Tm values, the specificity of all RPA primers and probes was assessed using the Blastn tool of the NCBI database, and finally three sets of primers and probe sets as shown in table 4 were obtained by screening.
TABLE 4 three sets of multiplex RPA primers and probe sets obtained by screening
RPA isothermal amplification was performed according to the method provided in example 1 using the three sets of primers and probes described in Table 4, respectively, and then detected by LFD chromatographic test strips, wherein the mcr-1, blaNDM-1 and tet (X4) contents of the samples to be detected were all 10 2 The magnitude of the copies/. Mu.L and the results of the test are shown in Table 5.
TABLE 5 influence of different primer and Probe sets on the RPA-LFD test results
As can be seen from Table 5, when screening different primers and probes for multiplex RPA amplification, there is a great influence on simultaneous detection of three drug-resistant genes in the amplified products, and when the first or second set of primers and probes are used, the work efficiency of mcr-1 and tet (X4) gene amplification primers and probes is insufficient, leadingIn the case of multiplex reaction, 10 2 The mcr-1 and tet (X4) genes at the concentration of copies/. Mu.L were not detected and did not meet the detection requirements. The third group of multiple RPA amplification primers and probes can realize the simultaneous, rapid and accurate detection of three drug-resistant genes, and the quantitative result is close to the known concentration, so that the third group of multiple RPA amplification primers and probes are selected for subsequent experiments.
Example 3 screening of primer and Probe concentrations
In this example, the RPA-LFD method provided in example 1 was used to detect mcr-1, blaNDM-1 and tet (X4), and isothermal amplification was performed using the primer and probe sets shown in Table 1, wherein the primer and probe concentrations were each isothermal amplified using the following 7 concentrations:
1 st: the concentration of the primer and probe of the mcr-1 gene was 0.15. Mu.M/0.06. Mu.M, the concentration of the primer and probe of the blaNDM-1 gene was 0.15. Mu.M/0.06. Mu.M, and the concentration of the primer and probe of the tet (X4) gene was 0.15. Mu.M/0.06. Mu.M;
2 nd: the concentration of the primer and probe for the mcr-1 gene was 0.30. Mu.M/0.09. Mu.M, the concentration of the primer and probe for the blaNDM-1 gene was 0.30. Mu.M/0.09. Mu.M, and the concentration of the primer and probe for the tet (X4) gene was 0.30. Mu.M/0.09. Mu.M;
3 rd: the concentration of the primer and probe for the mcr-1 gene was 0.42. Mu.M/0.12. Mu.M, the concentration of the primer and probe for the blaNDM-1 gene was 0.30. Mu.M/0.09. Mu.M, and the concentration of the primer and probe for the tet (X4) gene was 0.15. Mu.M/0.06. Mu.M;
4 th: the concentration of the primer and probe for the mcr-1 gene was 0.30. Mu.M/0.09. Mu.M, the concentration of the primer and probe for the blaNDM-1 gene was 0.42. Mu.M/0.12. Mu.M, and the concentration of the primer and probe for the tet (X4) gene was 0.15. Mu.M/0.06. Mu.M;
5 th: the concentration of the primer and probe for the mcr-1 gene was 0.15. Mu.M/0.06. Mu.M, the concentration of the primer and probe for the blaNDM-1 gene was 0.30. Mu.M/0.09. Mu.M, and the concentration of the primer and probe for the tet (X4) gene was 0.42. Mu.M/0.12. Mu.M;
6 th: the concentration of the primer and probe for the mcr-1 gene was 0.30. Mu.M/0.09. Mu.M, the concentration of the primer and probe for the blaNDM-1 gene was 0.42. Mu.M/0.12. Mu.M, and the concentration of the primer and probe for the tet (X4) gene was 0.30. Mu.M/0.09. Mu.M;
7 th: the concentration of the primer and probe for the mcr-1 gene was 0.42. Mu.M/0.12. Mu.M, the concentration of the primer and probe for the blaNDM-1 gene was 0.42. Mu.M/0.12. Mu.M, and the concentration of the primer and probe for the tet (X4) gene was 0.42. Mu.M/0.12. Mu.M.
The concentrations of the 7 primers and the probe are adopted to carry out RPA isothermal amplification, and the influence of different concentrations of the primers and the probe on the detection results of three drug resistance genes is examined. The mcr-1, blaNDM-1 and tet (X4) contents of the sample to be tested were all known to be 10 3 On the order of copies/. Mu.L. The reagent strips of the RPA-LFD test result are shown in FIG. 3, and the T/C value results are shown in FIG. 4.
As can be seen from fig. 3 and 4, there is a tendency that the detected T/C value increases with increasing concentration of the primer and probe, but the T/C value cannot be further increased and the decrease occurs when the concentration of the primer and probe is at the 7 th highest concentration, so that the primer and probe concentrations are preferably at the 6 th concentration: the concentration of the primer and probe for the mcr-1 gene was 0.30. Mu.M/0.09. Mu.M, the concentration of the primer and probe for the blaNDM-1 gene was 0.42. Mu.M/0.12. Mu.M, and the concentration of the primer and probe for the tet (X4) gene was 0.30. Mu.M/0.09. Mu.M, wherein the concentrations of the forward primer and the reverse primer were identical.
EXAMPLE 4 screening of magnesium acetate solution concentration
In this example, the RPA-LFD method provided in example 1 was used to detect mcr-1, blaNDM-1 and tet (X4), isothermal amplification was performed using the primer and probe set shown in Table 1, the concentration of the primer and probe for the mcr-1 gene was 0.30. Mu.M/0.09. Mu.M, the concentration of the primer and probe for the blaNDM-1 gene was 0.42. Mu.M/0.12. Mu.M, and the concentration of the primer and probe for the tet (X4) gene was 0.30. Mu.M/0.09. Mu.M, wherein the concentration of the magnesium acetate solution was 7 such as 0, 2.8, 5.6, 8.4, 11.2, 14, and 16.8mM, respectively, and the results of the RPA-LFD test were shown in FIG. 5, and the results of the T/C values are shown in FIG. 6.
As can be seen from FIGS. 5 and 6, the detected T/C value tended to rise and fall as the concentration of the magnesium acetate solution increased, and the detected T/C values were highest for mcr-1, blaNDM-1 and tet (X4) when the concentration of the magnesium acetate solution was 14mM (6 th). Thus, the concentration of the magnesium acetate solution is preferably 14mM.
EXAMPLE 5 screening of RPA isothermal amplification temperatures
In this example, the RPA isothermal amplification was performed by using the RPA-LFD method provided in example 1, with the primer and probe set shown in Table 1 at 0.30. Mu.M/0.09. Mu.M for the mcr-1 gene, 0.42. Mu.M/0.12. Mu.M for the blaNDM-1 gene, 0.30. Mu.M/0.09. Mu.M for the tet (X4) gene, and 14mM for the magnesium acetate solution, and the RPA isothermal amplification was performed at 6 temperatures of 30, 35, 37, 39, 45, and 50℃respectively, and the results of the RPA-LFD detection were shown in FIG. 7 and the results of the T/C values are shown in FIG. 8.
As can be seen from FIGS. 7 and 8, the detected T/C value tends to rise and fall with the rise of the RPA isothermal amplification temperature, and the detected T/C values of mcr-1, blaNDM-1 and tet (X4) reach the highest when the RPA isothermal amplification temperature is 37 ℃. Therefore, the RPA isothermal amplification temperature is preferably 37 ℃.
EXAMPLE 6 screening of RPA isothermal amplification time
In this example, the RPA isothermal amplification was performed using the RPA-LFD method provided in example 1, with the primer and probe set shown in Table 1, the concentration of the primer and probe of the mcr-1 gene being 0.30. Mu.M/0.09. Mu.M, the concentration of the primer and probe of the blaNDM-1 gene being 0.42. Mu.M/0.12. Mu.M, the concentration of the primer and probe of the tet (X4) gene being 0.30. Mu.M/0.09. Mu.M, and the concentration of the magnesium acetate solution being 14mM, and the RPA isothermal amplification being performed at 37℃for an isothermal amplification time of 0, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30min, and the results of the RPA-LFD assay being shown in FIG. 9, and the results of the T/C values are shown in FIG. 10.
As can be seen from FIGS. 9 and 10, lines T1, T2, T3 are all clearly visible after 5min of amplification. The detected T/C value steadily rises along with the extension of the RPA isothermal amplification time, but after the RPA isothermal amplification time is prolonged to 20min, the T/C values detected by mcr-1, blaNDM-1 and tet (X4) basically remain stable and have no significant difference from the T/C values detected at 25min and 30min. Therefore, in order to shorten the detection reagent, it is preferable that the isothermal amplification time of RPA is 20 minutes.
Example 7 verification of RPA-LFD detection method
In this example, triple RPA-LFD analysis and conventional PCR methods were used to analyze drug resistance genes carried by different Enterobacteriaceae strains. The 19 different Enterobacteriaceae strains are shown in Table 6, and include E.coli 14 strain, fei Kusen E.coli 1 strain, klebsiella pneumoniae 2 strain, salmonella 2 strain, 3 recombinant E.coli strains (Top 10-pUC-mcr-1, top10-pUC-blaNDM-1, top10-pUC-tet (X4)) containing standard plasmids carrying mcr-1, blaNDM-1 or tet (X4) genes, and an original E.coli strain E.coli ATCC25922 not carrying drug resistance genes, together 23 strains, the detailed information of each strain being shown in Table 6, as reference strains for optimizing the reaction system and analytical sensitivity.
TABLE 6 detection results
The drug resistance genes carried by 19 enterobacteriaceae strains were separated by triple RPA-LFD analysis and conventional PCR analysis, and the analysis results are shown in Table 7.
TABLE 7 analysis of the applications of the triple rpa-lfd method and pcr method
As can be seen from Table 7, both the triple RPA-LFD assay and the conventional PCR method can be used to detect 6 mcr-1 positive strains, 3 blaNDM-1 positive strains, 6 tet (X4) positive strains, and 4 mcr-1 and blaNDM-1 positive strains. The results of the target resistance gene detection by the two methods are consistent. But triple RPA-LFD analysis, including sample pretreatment, RPA amplification and LFD lateral flow, can be performed simultaneously for three genes, all within 40 minutes. In contrast, the PCR method, including sample pretreatment, PCR amplification and agarose gel electrophoresis, requires at least more than 3 hours, so the triple RPA-LFD analysis method is simpler, requires shorter time, and can be accurately quantified.
EXAMPLE 8 specific analysis of RPA-LFD to detect multiple drug resistance genes
In this example, in order to verify the specificity of the RPA-LFD method for detecting mcr-1, blaNDM-1 and tet (X4), the following nine bacterial liquids were used, respectively, and the detection was performed according to the method provided in example 1: 1. standard recombinant escherichia coli strains (Top 10-pUC-mcr-1+Top10-pUC-blaNDM-1+Top10pUC-tet (X4)) containing three drug resistance genes of mcr-1, blaNDM-1 and tet (X4) simultaneously; 2. recombinant escherichia coli strain (Top 10-pUC-mcr-1+Top10-pUC-blaNDM-1) containing mcr-1 and blaNDM-1 drug resistance genes simultaneously; 3. recombinant escherichia coli strain (Top 10-pUC-mcr-1+Top10pUC-tet (X4)) containing both mcr-1 and tet (X4) resistance genes; 4. recombinant escherichia coli strain (Top 10-pUC-blaNDM-1+Top10pUC-tet (X4)) containing two drug-resistant genes simultaneously; 5. a bacterial solution of recombinant E.coli strain (Top 10-pUC-mcr-1) containing only mcr-1; 6. a bacterial solution of a recombinant E.coli strain (Top 10-pUC-blaNDM-1) containing only blaNDM-1; 7. a bacterial solution of a recombinant E.coli strain (Top 10-pUC-tet (X4)) containing only tet (X4); 8. standard E.coli strain E.coli ATCC25922, which does not contain three drug resistance genes mcr-1, blaNDM-1 and tet (X4); 9. negative control. The detection results are shown in FIG. 11.
From FIG. 11, it can be seen that the LFD test strip detection result is completely consistent with the drug resistance gene actually contained in the recombinant E.coli strain of the member, and the triple RPA-LFD detection method provided by the invention has very good detection specificity on the target gene.
EXAMPLE 9 sensitivity analysis of RPA-LFD detection of multiple drug resistance genes
In order to evaluate the sensitivity of the RPA-LFD method for detecting mcr-1, blaNDM-1 and tet (X4), the bacterial solutions were diluted to specific concentrations, the plasmid concentrations in the bacterial solutions were measured, and the copy numbers of the plasmids were calculated to be 10, respectively 0 、10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 The concentration of copies/. Mu.L was measured by the method provided in example 1, and the measurement results are shown in FIG. 12.
As can be seen from FIG. 12, when the concentration of the drug-resistant gene is 10 1 When the samples are per mu L, the T value intensity can be successfully detected by the T1, T2 and T3 lines, so that the detection limit of the RPA-LFD method provided by the invention for detecting mcr-1, blaNDM-1 and tet (X4) is 10 samples per mu L.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.

Claims (10)

1. The primer combination for simultaneously detecting three drug resistance genes based on RPA-LFD is characterized by comprising a forward primer, a reverse primer and a probe, wherein the sequences of the primer combination are shown in the following table:
drug resistance gene Forward primer Reverse primer Probe with a probe tip mcr-1 SEQ ID NO：1 SEQ ID NO：2 SEQ ID NO：3 blaNDM-1 SEQ ID NO：4 SEQ ID NO：5 SEQ ID NO：6 tet(X4) SEQ ID NO：7 SEQ ID NO：8 SEQ ID NO：9
The three drug resistant genes are mcr-1, blaNDM-1 and tet (X4).
2. The primer combination of claim 1, wherein the 5' end of the probes of mcr-1, blaNDM-1, tet (X4) are modified with different groups capable of distinguishing three drug-resistant genes, respectively, the groups being biotin, cy5 or TAMRA; performing closed modification at the 3' end, and modifying a tetrahydrofuran abasic site in the probe sequence; digoxin is labeled at the 5' end of the reverse primer of mcr-1, blaNDM-1, tet (X4).
3. A kit for simultaneous detection of three drug-resistance genes, comprising the primer and probe combination of claim 1 or 2.
4. The kit according to claim 3, wherein the mcr-1, blaNDM-1 and tet (X4) have a primer concentration of 0.15 to 0.42. Mu.M and a probe concentration of 0.06 to 0.12. Mu.M.
5. The kit of claim 4, further comprising an RPA reaction dry powder and a test strip; the test strip is coated with Digoxin antibody, biotin antibody, cy5 antibody and TAMRA antibody.
6. The kit of claim 5, wherein the test strip is coated with Digoxin antibody, and is provided with a control line C line and three detection lines T1, T2 and T3, wherein the C line is coated with goat anti-mouse secondary antibody, and the T1, T2 and T3 are respectively coated with biotin antibody, cy5 antibody and TAMRA antibody, which respectively correspond to detection mcr-1, blaNDM-1 and tet (X4) genes.
7. The kit according to claim 6, further comprising a magnesium acetate solution having a concentration of 5 to 16.8mM.
8. A method for detecting three drug resistance genes using the kit according to any one of claims 3 to 7, comprising the steps of:
(1) Extracting nucleic acid to be detected;
(2) Adding RPA reaction dry powder, nucleic acid to be detected and magnesium acetate solution into a reaction tube;
(3) Isothermal amplification;
(4) And detecting by using a test strip, and judging a detection result.
9. The method according to claim 8, wherein the step (1) of extracting the nucleic acid to be tested is performed by extracting DNA by a boiling method.
10. The method of claim 8, wherein the isothermal amplification in step (3) is performed at a temperature of 25 to 43 ℃ for a period of 10 to 30 minutes.
CN202211384851.1A 2022-11-07 2022-11-07 Kit for simultaneously detecting multiple key drug resistance genes based on RPA-LFD and application thereof Pending CN116555449A (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN117417989A (en) * 2023-11-17 2024-01-19 河北省畜牧兽医研究所 Multiplex fluorescence quantitative PCR kit and method for detecting drug-resistant genes

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117417989A (en) * 2023-11-17 2024-01-19 河北省畜牧兽医研究所 Multiplex fluorescence quantitative PCR kit and method for detecting drug-resistant genes
CN117417989B (en) * 2023-11-17 2024-05-14 河北省畜牧兽医研究所 Multiplex fluorescence quantitative PCR kit and method for detecting drug-resistant genes

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