CN111440886A - Primer group, kit and detection method for rapidly detecting carbapenemase gene - Google Patents
Primer group, kit and detection method for rapidly detecting carbapenemase gene Download PDFInfo
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Abstract
The invention discloses a primer group, a kit and a detection method for rapidly detecting carbapenemase genes, wherein the primer group comprises an amplification primer pair and a molecular beacon probe for detecting carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP of enterobacter, and the nucleotide sequences are shown as SEQ ID NO 1-SEQ ID NO 15. The kit disclosed by the invention only needs to finish the common carbapenemase drug-resistant gene detection of five enterobacteriaceae within 2 hours by one-time sample adding, one reaction tube and one reaction system, has the advantages of rapidness, high efficiency, accuracy and the like, greatly shortens the detection time, saves the detection cost and improves the detection efficiency; compared with the existing probe method multiple PCR, the method reduces the design of primers and the interference among the primers, and can reduce the occurrence of false positive/false negative.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a primer group, a kit and a detection method for rapidly detecting carbapenemase genes.
Background
Bacterial resistance has become a global concern and infections caused by resistant bacteria have become a new challenge for clinical anti-infective therapy, a major threat to human health and life today. Enterobacteriaceae bacteria are widely distributed and closely related to humans, and Enterobacteriaceae are important nosocomial infectious bacteria, Carbapenem antibiotics are major antibiotics for treating Enterobacteriaceae infection, but in recent years, with the use of large amounts of Carbapenem antibacterial agents, Carbapenem drug-resistant Enterobacteriaceae (CRE) have been clinically developed. At present, the carbapenemase resistance genes which are clinically common mainly comprise KPC, NDM, OXA48, VIM and IMP. Although related kits for carbapenemase drug resistance gene detection exist in the market, most of them are used for singly detecting one of drug resistance genes such as KPC, NDM, OXA48, VIM and IMP.
The method is characterized in that the CRE bacteria is identified as soon as possible and drug sensitivity is reported in time, so that the CRE bacteria is important for guiding clinical use of reasonable antibiotics, the morbidity and the mortality can be effectively reduced, the culture identification is a traditional means for diagnosing pathogenic infection and has important significance for clinical early diagnosis and treatment, the overall time consumption of a culture identification process generally adopted in domestic laboratories at present is about 48-72 hours, the time consumption of the method is long, and the requirement of clinical quick identification and treatment on the CRE bacterial strain infection cannot be met.
Based on the molecular beacon technology invented by the Public Health Research Institute (PHRI) of the American Rutgers university, multiple genes can be simultaneously detected in the same homogeneous system by specifically recognizing nucleic acid sequences, and the method has the advantages of simplicity, rapidness, specificity and sensitivity. And a series of rapid detection and diagnosis platforms have been established in the aspects of clinical microbial infection and drug resistance, and comprise: drug-resistant mycobacterium tuberculosis, staphylococcus aureus identification and drug-resistant genes, enterobacteriaceae bacteria identification and carbapenem drug-resistant related gene identification. However, no kit for directly detecting carbapenemases by using molecular beacon technology has been available so far, and particularly no kit for simultaneously detecting genes KPC, NDM, OXA48, VIM and IMP at one time has been available.
Disclosure of Invention
The invention aims to provide a primer group, a kit and a detection method for rapidly detecting carbapenemase genes, wherein a molecular beacon technology is used for designing a specific primer, a melting curve method is used for carrying out quintuple fluorescence quantitative PCR (polymerase chain reaction) by the molecular beacon technology, and the rapid detection of carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP of enterobacter is realized. The method saves sample adding time and experiment cost, has the advantage of quickly and effectively detecting a plurality of genes at one time, and can provide effective data for clinical detection in time.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a primer group for rapidly detecting carbapenemase genes, which comprises an amplification primer pair and a molecular beacon probe, wherein the amplification primer pair is used for detecting carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP of enterobacter;
wherein, the primer group of the KPC gene is as follows:
the nucleotide sequence of KPC-F is shown in SEQ ID NO. 1;
the nucleotide sequence of KPC-R is shown in SEQ ID NO. 2;
the nucleotide sequence of the molecular beacon probe KPC-probe is shown in SEQ ID NO. 3;
the primer group of the NDM gene is as follows:
the nucleotide sequence of NDM-F is shown in SEQ ID NO. 4;
the nucleotide sequence of NDM-R is shown in SEQ ID NO. 5;
the nucleotide sequence of the molecular beacon probe NDM-probe is shown as SEQ ID NO. 6;
the primer set of the OXA48 gene is as follows:
the nucleotide sequence of the OXA48-F is shown as SEQ ID NO. 7;
the nucleotide sequence of the OXA48-R is shown as SEQ ID NO. 8;
the nucleotide sequence of the molecular beacon probe OXA48-probe is shown as SEQ ID NO. 9;
the primer group of VIM gene is as follows:
the nucleotide sequence of VIM-F is shown in SEQ ID NO. 10;
the nucleotide sequence of VIM-R is shown in SEQ ID NO. 11;
the nucleotide sequence of the molecular beacon probe VIM-probe is shown as SEQ ID NO. 12;
the primer group of IMP gene is as follows:
the nucleotide sequence of IMP-F is shown as SEQ ID NO. 13;
the nucleotide sequence of IMP-R is shown in SEQ ID NO. 14;
the nucleotide sequence of the molecular beacon probe IMP-MB is shown in SEQ ID NO. 15.
The invention also provides a kit for rapidly detecting the carbapenemase gene, which comprises the primer group for rapidly detecting the carbapenemase gene.
Preferably, the method further comprises the following steps: ex Taq enzyme, dNTP mix, Mg2+RNaseH, DNA template and SYBRGreenI; the concentration of the DNA template is 20-100 ng/ml.
Preferably, every 25 mu L PCR amplification reaction system contains 2U/. mu. L Ex Taq enzyme 1 mu. L, 2.5mM dNTPMixture 4 mu L and 1.5mM Mg 2+3 mu L, 2.5U/. mu. L RNaseH 1.5. mu. L I0.5. mu. L, wherein each primer and each molecular beacon probe in the primer group are respectively 0.2. mu. L and ddH2O was supplemented to 25. mu. L.
The invention also provides a method for detecting the carbapenemase drug-resistant gene of the enterobacteria by using the kit for non-diagnosis and treatment purposes, which is characterized by comprising the following steps:
step 1: extracting DNA of thalli in a sample to be detected;
step 2: taking the extracted DNA as a template, and carrying out quintuple fluorescent quantitative PCR by using the primer group; the concentration of the DNA template is 20-100 ng/ml;
and step 3: and performing melting curve analysis on the amplification products, and taking a positive quality control product as a control to identify the carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP of the enterobacter in the sample.
Preferably, the PCR reaction procedure in step 2 is: 95 ℃ for 10min, 95 ℃ for 10s, 58 ℃ for 15s, 72 ℃ for 20s, 45 cycles.
Preferably, the melting curve analysis procedure in step 3 is: denaturation at 95 deg.C for 1 min; the temperature rise range is 65-99 ℃, the temperature rise rate is 0.11 ℃/s, fluorescence signals are continuously collected for 5 times/DEG C, and melting curve analysis is carried out.
The invention discloses the following technical effects:
(1) the method for rapidly identifying the CRE strain is established by adopting a molecular beacon technology, designing a specific primer and jointly applying a real-time PCR technology, only one sample adding, one reaction tube and one reaction system are needed to finish the detection of the carbapenemase drug-resistant genes common to five enterobacteriaceae within 2 hours, and the method has the advantages of rapidness, high efficiency, accuracy and the like, greatly shortens the detection time, saves the detection cost and improves the detection efficiency.
(2) Compared with other multiplex PCR, such as common multiplex PCR, agarose gel electrophoresis is needed after amplification, and the products are distinguished by different lengths; after the reaction, the cover needs to be opened, so that the pollution is easily caused. For example, in the probe-based multiplex PCR, primers and probes need to be designed and synthesized simultaneously, and modifying groups of the taqman probe are expensive and easily interfere with each other. The multiple fluorescence quantitative PCR melting curve method adopting the molecular beacon technology is an economic, rapid and effective method, and the whole reaction process is totally closed and stable.
Drawings
FIG. 1 is a quintuple fluorescent quantitative PCR amplification curve of the present invention;
FIG. 2 shows the agarose gel electrophoresis detection of the amplification effect of each primer pair after the general PCR of the present invention.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
Composition of kit for rapidly detecting carbapenemase gene and detection method
1. Composition of the kit
(1) A primer group: amplification primer pairs and molecular beacon probes for detecting carbapenemase resistance genes KPC, NDM, OXA48, VIM and IMP of enterobacteria;
wherein, the primer group of the KPC gene is as follows:
KPC-F(SEQ ID NO:1)：CAGCTCATTCAAGGGCTTTC；
KPC-R(SEQ ID NO:2)：CGTCATGCCTGTTGTCAGAT；
molecular Beacon Probe KPC-Probe (SEQ ID NO: 3): cgcgaACACACCCATCCGTTACGGCA tcgcg, respectively;
the primer group of the NDM gene is as follows:
NDM-F(SEQ ID NO:4)：ATATCACCGTTGGGATCGAC；
NDM-R(SEQ ID NO:5)：TAGTGCTCAGTGTCGGCATC；
molecular Beacon Probe NDM-probe (SEQ ID NO: 6): cgcgaGCCTGATCAAGGACAGCAAGG tcgcg, respectively;
the primer set of the OXA48 gene is as follows:
OXA48-F(SEQ ID NO:7)：GTGGCATCGATTATCGGAAT；
OXA48-R(SEQ ID NO:8)：AGAGCACAACTACGCCCTGT；
molecular beacon probe OXA48-probe (SEQ ID NO: 9): cgcgaCAAGAAAACAAAAGTTGGAATGCtcgcg, respectively;
the primer group of VIM gene is as follows:
VIM-F(SEQ ID NO:10)：TCTACCCGTCCAATGGTCTC；
VIM-R(SEQ ID NO:11)：TTTCAATCTCCGCGAGAAGT；
molecular Beacon Probe VIM-Probe (SEQ ID NO: 12): cgcgaTGATTGATACAGCGTGGGGT tcgcg, respectively;
the primer group of IMP gene is as follows:
IMP-F(SEQ ID NO:13)：YGGAATAGAGTGGCTTAATTCTC；
IMP-R(SEQ ID NO:14)：TARCCAAACCACTACGTTATCT；
molecular beacon probe IMP-MB (SEQ ID NO: 15): cgcgaCCCACGTATGCATCTGAATTAACA tcgcg are provided.
(2) Process for preparing reagent kit
25 μ L PCR reactions were prepared (as shown in Table 1):
TABLE 125 μ L PCR reaction solution
2. Method for detecting drug-resistant gene of enterobacter carbapenemase by using kit
Step 1: extracting nucleic acid of a sample: extracting the DNA of the thallus in a sample to be detected by adopting a DNA extraction kit (purchased from Dalibao bioengineering Co., Ltd.);
the PCR reaction program is: 95 ℃ for 10min, 95 ℃ for 10s, 58 ℃ for 15s, 72 ℃ for 20s, 45 cycles.
And step 3: and performing melting curve analysis on the amplification products, and taking a positive quality control product as a control to identify the carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP of the enterobacter in the sample. The melting curve analysis program was: denaturation at 95 deg.C for 1 min; the temperature rise range is 65-99 ℃, the temperature rise rate is 0.11 ℃/s, fluorescence signals are continuously collected for 5 times/DEG C, and melting curve analysis is carried out.
Example 2
Method for rapidly detecting carbapenemase gene by using kit described in example 1
1. Sample(s)
Randomly collecting CRE strains of ST11 Klebsiella pneumoniae separated from clinical samples, respectively culturing the separated CRE strains for 24h at 37 ℃, extracting DNA by using the method described in example 1, screening corresponding positive samples by PCR through specific primers of five common carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP, sequencing and identifying, then selecting DNA samples of single carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP, and mixing the DNA samples to prepare the DNA samples containing the five common carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP. The clinical specimen used in the present example is a blood specimen, but is not limited thereto, and any respiratory tract specimen such as urine and sputum containing CRE strain of klebsiella pneumoniae type ST11 may be used as the clinical specimen.
Firstly, preparing PCR reaction solution according to table 1 in example 1, arranging 3 parallel reaction solutions, subpackaging the reaction solutions into 2m L reaction tubes, respectively adding a template to be detected, covering a tube cover, putting into a Roche L light cycler 96 fluorescent real-time PCR instrument, and carrying out PCR reaction by adopting the following reaction program of 95 ℃ for 10min, 95 ℃ for 10s, 58 ℃ for 15s, 72 ℃ for 20s and 45 cycles.
After the amplification is finished, performing melting curve detection, wherein the selected detection channels are FAM and HEX, and the melting process is 95 ℃ denaturation for 1 min; detecting the temperature rise range to 65-99 ℃ and the temperature rise rate to 0.11 ℃/s, continuously collecting fluorescence signals for 5 times/DEG C, and analyzing a melting curve.
As a result, it was found that: in the same reaction system, the carbapenemase drug-resistant gene detection common to five enterobacteriaceae is completed within 2 hours, and the carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP are detected by a primer designed by a molecular beacon technology through quintuple PCR, so that 5 amplification products can be accurately distinguished (as shown in figure 1).
In order to ensure the accuracy of the result, the invention also detects the amplification effect of each pair of primers by 1% agarose gel electrophoresis after the common PCR amplification. The results show that: each pair of primers can be used for carrying out independent amplification on different genes of the separated strains, no cross reaction exists, clear and bright bands can be generated (as shown in figure 2), and the detection result is consistent with the detection result by combining the double PCR with the melting curve method, so that the detection method disclosed by the invention is high in reliability.
Example 3
Sensitivity analysis of constant temperature amplification melting curve detection kit for CRE strain separated from clinical sample based on molecular beacon technology
1. Method of producing a composite material
Sample treatment, the CRE strain of Klebsiella pneumoniae type ST11 separated from clinical samples is adjusted to 1.8 × 10 concentration by physiological saline8CFU/m L, then gradient dilutedReleasing (diluting with 100 μ L bacterial solution +900 μ L physiological saline), centrifuging at 10000rpm, discarding supernatant, crushing on a shaker for 10min, adding supernatant 300 μ L into 96-deep-well plate, extracting DNA on nucleic acid extractor with 1.5 × 10%7CFU/mL-1.5×102DNA extracted from CFU/m L was amplified at 5. mu. L per concentration gradient as a template.
2. The assay was performed using the assay described in example 1. And observing the melting curve peak Tm value after the reaction is finished, and analyzing the detection effect of the kit and the lowest limit of the concentration of the template which can be detected.
3. The results show that the CRE strain separated from the clinical sample detected by the invention can effectively detect genes in strains with samples of different concentrations by using a kit, and the CRE strain with the concentration of 1.5 × 10 can be detected in 2 hours2The kit for rapidly detecting the carbapenemase gene has the advantages of rapidness, high efficiency and high sensitivity, and can play an important role in rapidly identifying clinically common carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (7)
1. A primer group for rapidly detecting carbapenemase genes, which is characterized by comprising an amplification primer pair and a molecular beacon probe, wherein the amplification primer pair is used for detecting carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP of enterobacter;
wherein, the primer group of the KPC gene is as follows:
the nucleotide sequence of KPC-F is shown in SEQ ID NO. 1;
the nucleotide sequence of KPC-R is shown in SEQ ID NO. 2;
the nucleotide sequence of the molecular beacon probe KPC-probe is shown in SEQ ID NO. 3;
the primer group of the NDM gene is as follows:
the nucleotide sequence of NDM-F is shown in SEQ ID NO. 4;
the nucleotide sequence of NDM-R is shown in SEQ ID NO. 5;
the nucleotide sequence of the molecular beacon probe NDM-probe is shown as SEQ ID NO. 6;
the primer set of the OXA48 gene is as follows:
the nucleotide sequence of the OXA48-F is shown as SEQ ID NO. 7;
the nucleotide sequence of the OXA48-R is shown as SEQ ID NO. 8;
the nucleotide sequence of the molecular beacon probe OXA48-probe is shown as SEQ ID NO. 9;
the primer group of VIM gene is as follows:
the nucleotide sequence of VIM-F is shown in SEQ ID NO. 10;
the nucleotide sequence of VIM-R is shown in SEQ ID NO. 11;
the nucleotide sequence of the molecular beacon probe VIM-probe is shown as SEQ ID NO. 12;
the primer group of IMP gene is as follows:
the nucleotide sequence of IMP-F is shown as SEQ ID NO. 13;
the nucleotide sequence of IMP-R is shown in SEQ ID NO. 14;
the nucleotide sequence of the molecular beacon probe IMP-probe is shown in SEQ ID NO. 15.
2. A kit for rapidly detecting carbapenemase gene, which comprises the primer set for rapidly detecting carbapenemase gene of claim 1.
3. The kit for rapidly detecting carbapenemase gene of claim 2, which further comprises: ExTaq enzyme, dNTP mix, Mg2+RNaseH, DNA template and SYBRGreen I; the concentration of the DNA template is 20-100 ng/ml.
4. The kit for rapidly detecting carbapenemase gene according to claim 3, which comprisesIs characterized in that every 25 mu L PCR amplification reaction system comprises 2U/mu L Ex Taq enzyme 1 mu L, 2.5mM dNTP mix 4 mu L and 1.5mM Mg2+3 mu L, 2.5U/. mu. L RNaseH 1.5. mu. L I0.5. mu. L, wherein each primer and each molecular beacon probe in the primer group are respectively 0.2. mu. L and ddH2O was supplemented to 25. mu. L.
5. A method for detecting carbapenemase resistance gene of enterobacteriaceae by the kit of claim 2 for non-diagnostic and therapeutic purposes, which comprises the steps of:
step 1: extracting DNA of thalli in a sample to be detected;
step 2: taking the extracted DNA as a template, and carrying out quintuple fluorescent quantitative PCR by using the primer group; the concentration of the DNA template is 20-100 ng/ml;
and step 3: and performing melting curve analysis on the amplification products, and taking a positive quality control product as a control to identify the carbapenemase drug-resistant genes KPC, NDM, OXA48, VIM and IMP of the enterobacter in the sample.
6. The method for detecting carbapenemase resistance gene of Enterobacter according to the non-diagnostic and therapeutic kit of claim 5, wherein the PCR reaction program in step 2 is: 95 ℃ for 10min, 95 ℃ for 10s, 58 ℃ for 15s, 72 ℃ for 20s, 45 cycles.
7. The method for detecting carbapenemase resistance gene of Enterobacter according to claim 5, wherein the melting curve analysis program in step 3 is: denaturation at 95 deg.C for 1 min; the temperature rise range is 65-99 ℃, the temperature rise rate is 0.11 ℃/s, fluorescence signals are continuously collected for 5 times/DEG C, and melting curve analysis is carried out.
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