CN110904199A - Primer, method and kit for detecting carbapenemase blaKPC gene - Google Patents
Primer, method and kit for detecting carbapenemase blaKPC gene Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and particularly relates to a primer, a method and a kit for detecting carbapenemase blaKPC gene subtype. The invention provides a group of primers capable of specifically amplifying all gene subtypes of carbapenemase blaKPC; and designs a corresponding detection method and a corresponding kit on the basis of the method. The kit and the method for detecting the carbapenemase blaKPC gene subtype have the characteristics of rapidness, accuracy, simple operation and the like, and can cover all gene subtypes of a blaKPC gene family; the detection specificity is strong, and the detection has no cross reaction with other non-target genes or human genes; the sensitivity is high, and the minimum detection limit determined according to the copy number of the plasmid is 100copies per reaction. The invention can be used for in vitro quantitative detection of all genotypes of the carbapenemase blaKPC family, provides a theoretical basis for clinical determination of anti-infection, and provides a reference method for the epidemiological study of carbapenemase molecules.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer, a method and a kit for detecting a carbapenemase blaKPC gene.
Background
Carbapenem antibiotics (Carbapenems) belong to β -members of the lactam family, have high stability to plasmid-mediated Extended spectrum β -lactamases (Extended specturn β -lactames, ESBLs), chromosomes and plasmid-mediated cephalosporins, and have been considered as important methods for emergency treatment of drug-resistant disorders, however, with the recent widespread use of such antibiotics, there are more and more carbapenem antibiotic-resistant strains clinically appearing, including acinetobacter baumannii, pseudomonas aeruginosa, and recently reported bacteria.
The detection method for carbapenemase at present mainly comprises the following two aspects: the phenotypic identification method based on the hydrolysis of carbapenem antibiotics by the enzyme; the other is a molecular biological detection method based on carbapenemase coding genes. Phenotypic identification methods include Modified Hodge Test (MHT), carbapenemase activity inhibition Test, and chromogenic medium isolation culture of carbapenem-containing antibiotics, as recommended by the clinical laboratory standards Committee (CLSI). The methods all depend on isolated culture of strains, are complex to operate, take long time and cannot define the molecular epidemiological characteristics of the strains. The molecular biological detection method comprises a gene chip detection method based on a hybridization technology, a single PCR based on nucleic acid amplification, an LAMP technology, a multiplex PCR and real-time fluorescent quantitative PCR technology and a second-generation sequencing technology. The gene chip method has the advantages of high speed, high automation degree, high sensitivity and specificity and the like; however, this method is only suitable for a limited number of known genotypes, and cannot detect genotypes and gene subtypes of emerging enzymes, and the cost of instruments and equipment and reagents is high, and ordinary laboratories cannot be developed as routine work. The traditional single PCR combined agarose gel electrophoresis and gene sequencing technology is the gold standard for determining the carbapenemase genotype at present, but the method has large workload, is easy to cause PCR product pollution due to uncovering detection, has false positive, and is not suitable for large-scale clinical detection and epidemiological investigation. The traditional real-time fluorescent quantitative PCR or LAMP technology can judge the existence and the content of the genotype to be detected through an amplification curve generated by fluorescence change in the amplification process, but the length of an amplification fragment is generally limited to about 200bp, and the method is only limited to detect a limited number of known genotypes and cannot cover all genotypes or gene subtypes of the same family. Sequencing of the whole bacterial genome or plasmid can intuitively research the genotype and position of carbapenemase gene and find new genotype. However, the method is complex to operate, expensive, not suitable for large-scale clinical specimen detection and limited to the scientific research stage of a laboratory at present.
Disclosure of Invention
The invention aims to solve at least one technical problem in the prior art, and provides a primer, a method and a kit which are real-time, accurate and rapid and can detect all gene subtypes of a carbapenemase blaKPC gene family.
Therefore, an object of the present invention is to provide a primer for detecting a carbapenemase blaKPC gene subtype.
Another object of the present invention is to provide a kit for detecting a carbapenemase blaKPC gene subtype.
Still another object of the present invention is to provide a method for detecting a carbapenemase blaKPC gene subtype.
The invention also aims to provide application of the primer or the kit in detecting drug-resistant bacteria carrying blaKPC genes.
The technical scheme adopted by the invention is as follows.
The primer for detecting the carbapenemase blaKPC gene subtype comprises the following sequences:
KPC-F3：GAATATCGTTGATGTCACTG；
KPC-R3：AGATGATTTTCAGAGCCTTA。
in some embodiments of the invention, the primers described above may be synthesized by means conventional in the art.
Further, the carbapenemase blaKPC gene subtypes include: blaKPC-1, blaKPC-2, blaKPC-3, blaKPC-4, blaKPC-5, blaKPC-6, blaKPC-7, blaKPC-8, blaKPC-9, blaKPC-10, blaKPC-11, blaKPC-12, blaKPC-13, blaKPC-14, blaKPC-15, blaKPC-16, blaKPC-17, blaKPC-18, blaKPC-19, blaKPC-20, blaKPC-21, blaKPC-22, blaKPC-23, blaKPC-24, blaKPC-25, blaC-26, blaC-27, blaC-28, blaC-29, KPaC-30, blaKPaC-31, blaKPaKPC-32, blaKPC-33, blaKPC-35, blaKPC-33, blaKPC-35, blaKPC-32, blaKPC-19, blaKPC-20, blaKPC-21, blaKPC-23, blaKPC-C-23, blaKPC-24, blaKPC-32, bla, blaKPC-44, blaKPC-45, blaKPC-46.
In some embodiments of the invention, the primers are used to specifically amplify the entire sequence of a carbapenemase blaKPC-containing gene subtype.
The invention also provides application of the primer in preparing a reagent for detecting bacteria carrying the carbapenemase blaKPC gene subtype.
Further, the detecting includes:
1) extracting DNA from the sample;
2) carrying out PCR amplification reaction by using the extracted DNA as a template and the primer according to any one of claims 1 to 3;
3) and (3) carrying out result analysis on the carbapenemase blaKPC gene subtype in the sample to be detected.
Further, the PCR amplification reaction is a real-time quantitative PCR reaction.
These primers can be used in a method for detecting a carbapenemase blaKPC gene subtype in a sample whose presence indicates that a bacterium having carbapenemase blaKPC gene resistance is contained in the sample or in the preparation of a related reagent.
The invention also provides a kit for detecting the carbapenemase blaKPC gene subtype, which comprises the primer.
Further, the kit further comprises: positive control
Further, the positive control is a plasmid carrying the complete sequence of blaKPC.
Further, the plasmid was a cloned plasmid carrying the full sequence of blaKPC constructed using pET28a vector.
Furthermore, the consistency of the nucleic acid sequence of the plasmid and blaKPC-4 is 100%, and the nucleic acid sequence of GenBank accession numbers MK 816391.
Further, the kit further comprises: and (5) a negative control product.
Further, the negative control substance is sterilized double distilled water.
Further, the kit further comprises: 2 × SYBR Green PCR Master Mix, standards, and nuclease free deionized water.
Further, the 2 XSSYBR Green PCR Master Mix comprises Taq enzyme, dN (U) TP, 10 XSCRBuffer, SYBR Green fluorescent dye, MgCl2And a mixture of UNG enzyme.
Further, the standard substance is: pET28a-blaKPC positive clone plasmid, standard substance concentration is 10 separately8、107、106、105copies/ml。
The invention also provides a method for detecting the carbapenemase blaKPC gene subtype for non-diagnostic or therapeutic purposes, which comprises the following steps:
1) extracting DNA from the sample;
2) taking the extracted DNA as a template, and carrying out full-sequence fluorescent quantitative PCR amplification reaction by using the primer;
3) and (3) carrying out result analysis on the carbapenemase blaKPC gene subtype in the sample to be detected.
Further, in step 2), the reaction system of the fluorescent quantitative PCR amplification reaction is:
further, in step 2), the reaction procedure of the fluorescent quantitative PCR amplification reaction is: 2min at 50 ℃; 30s at 95 ℃; 10s at 95 ℃; 30s at 56 ℃ and 45s at 72 ℃ for 40 cycles; a melting curve step: the temperature of 65 ℃ is gradually increased to 98 ℃, the temperature is increased by 0.18 ℃ per second, and fluorescence is collected every 0.11 s.
Wherein, in the full sequence fluorescent quantitative PCR amplification reaction program, the program of 2min at 50 ℃ is used for pretreating UNG; the fluorescence was collected at 56 ℃ for 30 s.
The existing fluorescent quantitative PCR generally adopts a two-step procedure aiming at short segments, and the fluorescent quantitative PCR amplification of the carbapenemase blaKPC gene complete sequence is higher than the amplification of the short segments in difficulty degree, so the invention adopts a three-step PCR reaction procedure in research, prolongs the annealing and extending time, and ensures the full dissociation of DNA double strands and the synthesis of new double strands. In addition, a specific 2 XSSYBR Green PCR Master Mix reagent is selected, so that the success of amplification is ensured in many aspects.
Further, in step 3), the specific process of result analysis is as follows: when the Tm value of the melting curve of the sample to be detected is inconsistent with the Tm value of the positive control, the sample to be detected is negative; when the Tm value of the melting curve of the sample to be detected is consistent with the Tm value of the positive control, the amplification curve is S-shaped, and the Cp value is positive when displayed at 5-35; and drawing a standard curve according to the Cp value and the concentration of the standard product, substituting the Cp value of the sample to be detected into a standard curve formula, and calculating the concentration of the blaKPC family gene subtype contained in the sample to be detected.
The invention has the beneficial effects that:
the total length of the carbapenemase blaKPC gene is about 882bp, the known gene subtypes include 46, and mutation points of each gene subtype are positioned in the gene, the length of the amplification fragment of the existing fluorescent quantitative PCR technology is generally 150-250bp, only for the amplification of partial sequence of the gene, partial base mutation may occur in the amplification region of the primer, resulting in false negative result. Aiming at the complete sequence and flank of the carbapenemase blaKPC gene, the invention compares the complete sequence and flank with all gene subtypes of the blaKPC gene and designs a primer which can specifically amplify all the blaKPC subtypes; the kit and the method for detecting the carbapenemase blaKPC gene subtype established on the basis have the characteristics of rapidness, accuracy, simple operation and the like, and can cover all gene subtypes of a blaKPC gene family. The detection system has strong specificity and has no cross reaction with other non-target genes or human genes. The sensitivity is high, and the minimum detection limit determined according to the copy number of the plasmid is 100copies per reaction. The invention can be used for in vitro quantitative detection of all the gene types of KPC family in carbapenemase-resistant gram-negative bacillus, provides a theoretical basis for clinical selection of reasonable anti-infection measures, and provides a reference method for the epidemiological research of carbapenemase molecules.
Drawings
FIG. 1 is a diagram showing the alignment result of blaKPC full sequence Primer-BLAST;
FIG. 2 is a diagram showing the result of electrophoresis of PCR products under different annealing temperature amplification conditions;
FIG. 3 is a diagram showing the analysis of the positive result melting curve of blaKPC gene detected by full-sequence fluorescent quantitative PCR;
FIG. 4 is a diagram showing the result of specific detection by the kit;
FIG. 5 shows the full-sequence fluorescent quantitative PCR detection sensitivity analysis.
Detailed Description
The kit based on the full-sequence fluorescent quantitative PCR system can realize the purpose of quickly and accurately detecting all genotypes of the carbapenemase KPC family. Solves the technical problems of complex operation, time and labor consumption and capability of only detecting limited genotypes in the traditional phenotype identification method and the prior molecular biology method.
The experimental design flow of the invention is as follows:
firstly, the design and evaluation of the full sequence primer are as follows: the GenBank gene sequence database (http:// www.ncbi.nlm.nih.gov/nucleotide /) gene complete sequence and flank are adopted to be compared with ClustalX, and a conserved region is selected to design a specific primer aiming at whole gene amplification. All primers were analyzed by Oligo6, Primer Premier5.0, and NCBI Primer-BLAST software alignment to avoid potential non-specific amplified fragments. Through primer sequence analysis and PCR amplification experiment verification, a pair of specific primers for full-sequence amplification of the carbapenemase blaKPC gene are selected.
Secondly, specimen collection and DNA template preparation: collecting carbapenem antibiotic resistant strains, taking a single colony to prepare 2 McLee unit bacterial liquid, and taking 500 mu l to extract the whole genome DNA of the strains.
Furthermore, the optimization of PCR amplification conditions: each target gene was amplified using specific primers for the full sequence. After the PCR product is subjected to 1.5% agarose gel electrophoresis, the electrophoresis result is observed, the specificity of each pair of specific primers is preliminarily verified, and the optimal uniform annealing temperature of each pair of primers is determined by gradient PCR amplification.
Fourthly, establishment of a complete sequence fluorescent quantitative PCR detection system: and respectively using each pair of available specific primers after verification and DNA containing a known target gene as a template, and adopting a SYBR Green kit containing dUTG base groups to establish a full-sequence fluorescent quantitative PCR detection system.
Fifthly, the specificity analysis of the complete sequence fluorescent quantitative PCR detection system: and (3) verifying the specificity and the anti-interference capability of the multiple detection system by using the positive template of the target gene and DNA fragments of other non-target genes, including human genome DNA, other pathogens and DNA of drug-resistant genes.
Sixthly, the sensitivity analysis of the full-sequence fluorescent quantitative PCR detection system is as follows: preparing positive clone plasmid of target gene, calculating its copy number and performing gradient dilution by 10 times, verifying analysis sensitivity of multiple amplification system by target gene template gradient dilution, and determining the lowest detection limit of detection system.
And finally, the clinical applicability analysis of a complete sequence fluorescent quantitative PCR detection system: the detection system is adopted to screen clinically separated carbapenem drug-resistant strains, the distribution condition of each carbapenemase gene KPC family is observed, and compared with the strain phenotype identification combined traditional single PCR analysis result, the diagnosis sensitivity, the diagnosis specificity and the diagnosis goodness of fit (Kappa coefficient) of the detection system are analyzed, so as to evaluate the clinical practicability of the combined detection system.
The kit for detecting the carbapenemase blaKPC gene subtype is prepared according to the full-sequence fluorescent quantitative PCR detection system. The kit has strong specificity and has no cross reaction with other non-target genes or human genes. The sensitivity is high, and the minimum detection limit determined according to the copy number of the plasmid is 100copies per reaction. The invention can be used for in vitro quantitative detection of all the gene types of KPC family in carbapenemase-resistant gram-negative bacillus, provides a theoretical basis for clinical determination of anti-infection, and provides a reference method for the epidemiological study of carbapenemase molecules.
The technical solutions of the present invention are further described below with reference to the drawings and specific examples, but the present invention is not limited to these specific embodiments. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.
The nucleic acid extraction reagent is purchased from Guanghua silver pharmaceutical science and technology Co., Ltd, the general PCR amplification reagent is TakaraEx Taq purchased from Nipponbo, and the fluorescent quantitative PCR reagent is QuantiTect SYBR Green PCR Kit purchased from QIAGEN, Germany.
Example 1
Primer design
The complete gene sequence and flank of GenBank gene sequence database (http:// www.ncbi.nlm.nih.gov/nucleotide /) are compared with ClustalX, and a conserved region is selected to design a specific primer aiming at the complete gene amplification of carbapenemase blaKPC gene subtype. Primers were designed using NCBI Primer-BLAST, all primers were analyzed by Oligo6, PrimerPremier5.0, and NCBI Primer-BLAST software alignment to exclude potential non-specific amplified fragments. Under the condition of ensuring amplification of a blaKPC family complete sequence, a plurality of pairs of primers are designed, and the complete sequence and partial flanking sequence of the carbapenemase blaKPC gene are finally determined to be amplified by using only one pair of primers through GC content analysis, 3 'and 5' complementary sequence analysis, common PCR agarose experiment verification and other means, the Primer has good specificity and strong anti-interference capability, and the Primer-BLAST comparison result is shown in figure 1.
The primer sequence is as follows:
KPC-F3：GAATATCGTTGATGTCACTG；
KPC-R3：AGATGATTTTCAGAGCCTTA。
the amplified fragment was 910 bp.
The specific primer can amplify all the currently known genotypes of the carbapenemase blaKPC family, including: blaKPC-1, blaKPC-2, blaKPC-3, blaKPC-4, blaKPC-5, blaKPC-6, blaKPC-7, blaKPC-8, blaKPC-9, blaKPC-10, blaKPC-11, blaKPC-12, blaKPC-13, blaKPC-14, blaKPC-15, blaKPC-16, blaKPC-17, blaKPC-18, blaKPC-19, blaKPC-20, blaKPC-21, blaKPC-22, blaKPC-23, blaKPC-24, blaKPC-25, blaC-26, blaC-27, blaC-28, blaC-29, KPaC-30, blaKPaC-31, blaKPaKPC-32, blaKPC-33, blaKPC-35, blaKPC-33, blaKPC-35, blaKPC-32, blaKPC-19, blaKPC-20, blaKPC-21, blaKPC-23, blaKPC-C-23, blaKPC-24, blaKPC-32, bla, blaKPC-44, blaKPC-45, blaKPC-46.
Example 2
Positive template preparation and PCR amplification condition optimization
The positive strain containing the gene sequence of carbapenemase blaKPC gene subtype was collected, isolated and cultured in a selective medium containing 2mg/L imipenem, and a single colony was taken to prepare 2M cell suspension, 500. mu.L of the suspension was taken, the whole genome DNA of the strain was extracted as a template, and each target gene was amplified using the specific primer obtained in example 1 for the entire sequence.
The amplification system was as follows:
premix Ex Taq 12.5. mu.L (containing premixed Taq enzyme, dNTP, 10 XPCR Buffer, MgCl2) 10 μ M of specific upstream and downstream primers 1.5 μ L of each 1 μ L, DNA template (DNA content 100-300ng), complement 25 μ L of nuclease-free deionized water。
The gradient PCR amplification is adopted, and the reaction program is as follows: 30s at 94 ℃; annealing in a temperature gradient manner; 30s at 72 ℃ and 40 cycles. The annealing temperature was gradually increased from 54 ℃ to 60 ℃. After the PCR product was subjected to 1.5% agarose gel electrophoresis, the results are shown in FIG. 2, wherein bands 1 to 7 are PCR products obtained at annealing temperatures of 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃ and 60 ℃ respectively; the strip 8 is an NTC; the band M is DNA Marker. The electrophoresis result shows that a specific target band shows that the KPC F/R designed by the invention has good specificity, when the annealing temperature is 56 ℃, the PCR product amplification band is brightest, and the optimal annealing temperature is determined to be 56 ℃.
Example 3
Assembly of kit for detecting carbapenemase blaKPC gene subtype
The kit comprises the following components:
the QuantiTect SYBR Green PCR Kit is a premixed 2 XSSYBR Green PCR Master Mix comprising Taq enzyme, dN (U) TP, 10 XPCR Buffer, SYBR Green fluorescent dye, MgCl2A mixture of UNG enzyme;
negative control: sterilizing double distilled water;
positive control: the cloned plasmid carrying the full sequence of blaKPC constructed by pET28a vector, positive plasmid is sequenced, the consistency of nucleic acid sequence and blaKPC-4 is 100%, and GenBank accession numbers MK 816391.
And (3) standard substance: pET28a-blaKPC positive cloning plasmid 10 copies/. mu.L-106copies/μL。
The upstream and downstream specific primers KPC-F/-R are shown in example 1;
nuclease-free deionized water.
2. Method for using the above kit
(1) Configuring a full-sequence fluorescent quantitative PCR reaction system (shown in Table 1):
TABLE 1 complete sequence fluorescent quantitative PCR reaction system
Note: the amplification system is simultaneously provided with a positive control, a negative control, a standard substance and a blank control.
(2) And transferring the prepared reaction system into an eight-connection tube suitable for Roche 480, wherein each hole of the reaction system is 20 mu L, tightly covering the eight-connection tube with a cover, and performing instantaneous centrifugation to avoid reaction liquid remaining on the tube wall.
(3) And (3) placing the eight-connecting tube in a LighterCycler480 fluorescent quantitative PCR instrument for amplification, and carrying out melting curve analysis after the amplification reaction is finished.
The reaction procedure was as follows: 2min at 50 ℃ (UNG pre-treatment); 30s at 95 ℃; 10s at 95 ℃; 30s at 56 ℃ (fluorescence is collected), 45s at 72 ℃, and 40 cycles; a melting curve step: the temperature of 65 ℃ is gradually increased to 98 ℃, the temperature is increased by 0.18 ℃ per second, and fluorescence is collected every 0.11 s.
(4) And (4) judging a result: the detection result is jointly judged according to the Tm value of the melting curve and the Cp value of the amplification curve, and the Tm value of the melting curve of the sample to be detected is inconsistent with the Tm value of the positive control and is negative; the Tm value of the melting curve of the sample to be detected is consistent with the Tm value of a positive control (as shown in figure 3, the Tm value of the blaKPC melting curve can be seen in three groups of repeated experiments of the positive control, the Tm value is 92.39-92.57 ℃), the amplification curve is S-shaped, and the Cp value is positive when being displayed between 5-35; and drawing a standard curve according to the Cp value and the concentration of the standard substance, and calculating the concentration of the positive sample to be detected according to the Cp value of the sample to be detected.
Example 4
Method for detecting carbapenemase blaKPC gene subtype
1) Culturing blood of a patient infected with Klebsiella pneumoniae resistant to carbapenem antibiotics in a blood culture flask, and extracting 500. mu.L of positive culture fluid to extract DNA;
2) taking the extracted DNA as a template, and carrying out full-sequence fluorescent quantitative PCR amplification reaction to obtain an amplification product, wherein the reaction system is as follows:
the reaction procedure is as follows: 2min at 50 ℃ (UNG pre-treatment); 30s at 95 ℃; 10s at 95 ℃; 30s at 56 ℃ (fluorescence is collected), 45s at 72 ℃, and 40 cycles; a melting curve step: the temperature of 65 ℃ is gradually increased to 98 ℃, the temperature is increased by 0.18 ℃ per second, and fluorescence is collected every 0.11 s.
3) Analyzing the result of the carbapenemase blaKPC gene subtype in the sample to be detected;
the Tm value of the melting curve of the sample to be detected is consistent with the Tm value of the positive control, the amplification curve is S-shaped, and the Cp value is 25.12; and drawing a standard curve according to the Cp value and the concentration of the standard product, substituting the Cp value of the sample to be detected into a standard curve formula, and calculating that the concentration of the blaKPC family gene contained in the sample to be detected is 31600 copies.
Using the extracted DNA as a template, as described in example 2, using a common PCR reagent Premix Ex Taq for amplification, determining the size of the gene to be detected to be about 910bp by agarose gel electrophoresis of the PCR product, and comparing the nucleic acid sequence with the NCBI blastN database by first-generation sequencing, and finding that the gene to be detected is blaKPC-2 subtype.
Example 5
Example 3 specificity of the kit prepared
Mixing a positive template of a target gene with DNA fragments of other non-target genes, including human genome DNA, quality control strains of Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, Klebsiella pneumoniae ATCC70060 and Staphylococcus aureus ATCC25923, and DNA of other drug-resistant genes (carbapenemase genes blaNDM, blaVIM, blaIMP and blaOXA-23), and verifying the specificity and anti-interference capability of the kit;
the corresponding positive control provided in the kit was used as a positive control, sterilized double distilled water was used as a negative control, a reaction system was prepared according to table 1, and fluorescent quantitative detection was performed according to the reaction procedure in example 3, and the results of fluorescent quantitative PCR analysis showed that only the gene amplification curve of the positive control was in an S-shape, and the results are shown in fig. 4, in which fig. 4A, 4B, and 4C show that Cp values of blaKPC in three independent repeat tests were 20.87, 21.32, and 22.36, respectively. It is demonstrated that the kit of the invention has very good specificity for the carbapenemase blaKPC gene.
Example 6
Example 3 sensitivity of the kit prepared
A positive cloning plasmid for the target gene is prepared,calculating the copy number of the target gene, performing gradient dilution by 10 times, and verifying the analysis sensitivity of the multiple amplification system by using the target gene template gradient dilution, wherein the target gene concentration of each reaction tube is 106、105、104、103、102And 10copies, carrying out repeated experiments for each reaction system for multiple times, and determining the lowest detection limit of the detection system. The reaction system and the reaction procedure were carried out as in example 3.
The results are shown in FIG. 5, in which FIG. 5A is a 10-fold gradient dilution amplification curve of blaKPC plasmid; FIG. 5B is a 10 fold gradient dilution melting curve for blaKPC plasmid; FIG. 5C is a 10-fold gradient dilution standard curve for blaKPC plasmid. It can be seen that the detection lower limit of blaKPC plasmid can reach 100 copies/reaction system.
Example 7
Analysis of clinical suitability
The method is adopted to screen the clinically separated carbapenem drug-resistant strains, observe the distribution condition of each carbapenemase gene blaKPC family, compare the distribution condition with the strain phenotype identification combined traditional single PCR analysis result, and analyze the diagnosis sensitivity, the diagnosis specificity and the diagnosis goodness of fit (Kappa coefficient) of the detection system so as to evaluate the clinical practicability of the combined detection system.
The blaKPC gene was detected using 162 clinically isolated carbapenem-resistant gram-negative bacilli genomic DNAs as a template with the kit of example 3. As a result, as shown in table 2, it was found that 80 strains, 5 strains, 6 strains, and 71 strains, were resistant to the antibiotic phenotype and no carbapenemase blaKPC gene was detected, indicating that the results of the present invention were highly consistent with the drug susceptibility test, the sensitivity was 93.02% (80/86), the specificity was 93.42% (71/76), the positive predictive value was 94.11% (80/85), the negative predictive value was 92.20% (71/77), and the Kappa coefficient was 0.864.
TABLE 2 analysis of the efficacy of the full sequence fluorescent quantitative PCR and antibiotic sensitivity test for detecting carbapenem drug-resistant strains
In conclusion, the full-sequence fluorescent quantitative PCR detection kit established by the invention can detect all genotypes of the blaKPC family, has the advantages of rapidness, accuracy, high sensitivity, strong specificity, convenient implementation and the like, and the popularization and the application of the kit provide a basis for clinically and rapidly determining carbapenem drug-resistant bacteria infection, provide a new means for the molecular epidemiological research of carbapenem antibiotic drug resistance, and provide a new idea for the research of a bacterial drug-resistant mechanism.
It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.
Claims (9)
1. A primer for detecting carbapenemase blaKPC gene subtype is characterized by comprising the following sequences:
KPC-F3：GAATATCGTTGATGTCACTG；
KPC-R3：AGATGATTTTCAGAGCCTTA。
2. the primer according to claim 1, wherein the primer is used for specifically amplifying the whole sequence of a gene subtype containing carbapenemase blaKPC.
3. The primer according to claim 1, wherein said carbapenemase blaKPC gene subtype comprises: blaKPC-1, blaKPC-2, blaKPC-3, blaKPC-4, blaKPC-5, blaKPC-6, blaKPC-7, blaKPC-8, blaKPC-9, blaKPC-10, blaKPC-11, blaKPC-12, blaKPC-13, blaKPC-14, blaKPC-15, blaKPC-16, blaKPC-17, blaKPC-18, blaKPC-19, blaKPC-20, blaKPC-21, blaKPC-22, blaKPC-23, blaKPC-24, blaKPC-25, blaC-26, blaC-27, blaC-28, blaC-29, KPaC-30, blaKPaC-31, blaKPaKPC-32, blaKPC-33, blaKPC-35, blaKPC-33, blaKPC-35, blaKPC-32, blaKPC-19, blaKPC-20, blaKPC-21, blaKPC-23, blaKPC-C-23, blaKPC-24, blaKPC-32, bla, blaKPC-44, blaKPC-45, blaKPC-46.
4. Use of a primer according to any one of claims 1 to 3 for the preparation of a reagent for the detection of bacteria carrying the carbapenemase blaKPC gene subtype.
5. Use according to claim 4, characterized in that said detection comprises:
1) extracting DNA from the sample;
2) carrying out PCR amplification reaction by using the extracted DNA as a template and the primer according to any one of claims 1 to 3;
3) and (3) carrying out result analysis on the carbapenemase blaKPC gene subtype in the sample to be detected.
6. Use according to claim 5, wherein the PCR amplification reaction is a real-time quantitative PCR reaction.
7. A kit for detecting a carbapenemase blaKPC gene subtype, wherein the kit comprises the primer of any one of claims 1-3.
8. The kit of claim 7, further comprising: a negative control and a positive control.
9. A method for detecting a carbapenemase blaKPC gene subtype for non-diagnostic or therapeutic purposes, comprising the steps of:
1) extracting DNA from the sample;
2) carrying out PCR amplification reaction by using the extracted DNA as a template and the primer according to any one of claims 1 to 3;
3) and (3) carrying out result analysis on the carbapenemase blaKPC gene subtype in the sample to be detected.
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