CN111500751B - Detection method and kit for rapidly detecting high-virulence klebsiella pneumoniae - Google Patents
Detection method and kit for rapidly detecting high-virulence klebsiella pneumoniae Download PDFInfo
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Abstract
The invention discloses a detection method and a kit for rapidly detecting high-virulence klebsiella pneumoniae, wherein the method comprises the following steps: extracting strain DNA; placing the extracted strain DNA and the primer into a reaction solution for LAMP amplification reaction; the reaction solution comprises a primer, wherein the primer is respectively as follows: upstream outer primer F3:5'-TGGGGTTATTCTTTCGCT-3'; downstream outer primer B3:5'-TTTCCAAGCTTACTGCAATT-3'; upstream inner primer FIP:5'-CCagCAAAacAGCCTAAATACATTG-TGGGGAGTATCTTTGAGAGG-3'; downstream inner primer BIP:5'-TTGGGATACTGTGCTATTTTTCTCT-GGGAAGATGAGAAATACGAGC-3'; upstream loop primer LF:5'-CGCCTCCGTGATGAGGATG-3'; downstream loop primer LB:5'-GCAGAAAAGGGCTAGCGC-3'; after the LAMP amplification reaction is finished, the reaction solution is detected to judge whether the high-virulence klebsiella pneumoniae exists. According to the embodiment of the invention, 6 different areas of the high-virulence klebsiella pneumoniae peg-344 gene are amplified by designing 2 pairs of primers, so that a method for quickly, simply, conveniently and economically detecting the high-virulence klebsiella pneumoniae in sputum is established, and the method has good specificity characteristics.
Description
Technical Field
The invention relates to the technical field of microorganism detection, in particular to a detection method and a kit for rapidly detecting high-virulence klebsiella pneumoniae.
Background
Klebsiella pneumoniae is a common pathogenic bacterium for community-acquired and hospital-acquired infections. Klebsiella pneumoniae has thick capsular polysaccharide, and can effectively inhibit neutrophil phagocytosis and antibiotic action, thereby being more likely to cause pneumonia, blood flow infection, urinary tract infection and the like. Currently, infection caused by Klebsiella pneumoniae high virulent strains has become a worldwide infectious disease along with differences in capsular polysaccharide types and bacterial virulence transition. Compared with the classical Klebsiella pneumoniae (classic Klebsiella pneumoniae, cKP) with hospital-acquired infection, the hvKP has high death rate after infection and is accompanied by severe complications such as sepsis, metastatic infection and the like. In recent years, hvKP bacterial resistance and its relationship to bacterial fitness/virulence have been of worldwide interest. Especially, the emergence of carbapenem drug-resistant hvKP bacteria (CR-hvKP) brings serious challenges to clinical treatment and basic research.
Klebsiella pneumoniae is a common community-acquired and nosocomial infectious pathogen that can cause disseminated infections such as liver abscess, endophthalmitis, septicemia, and the like, and is mostly related to K1 and K2 capsular serotypes and high-mucus phenotypes, and is often defined as Klebsiella pneumoniae (hvKP) with high virulence. Over the last 30 years, it has been found that klebsiella pneumoniae has become the main causative agent of bacterial liver abscesses, especially klebsiella pneumoniae, which is highly virulent, is more common and is spreading as a new invasive syndrome. hvKP has a strong invasiveness and biofilm formation capacity, and the resulting infections include coeliac disease (community-acquired liver abscess, spleen abscess, spontaneous fine peritonitis), thoracic disease (pneumonia, empyema), endophthalmitis, central nervous system disease (meningitis), skeletal muscle and soft tissue infection, urinary tract infection, mixed infection, bacteremia, of which community-acquired liver abscess is the most common.
Detection of klebsiella pneumoniae with high virulence is an important means for judging the causative agents of disseminated infections such as liver abscess, endophthalmitis, septicemia and the like. Traditional methods for detecting the high-virulence klebsiella pneumoniae comprise colony morphology, wiredrawing test, nematode test, neutrophil anti-phagocytosis test, serum resistance test and the like, and the methods are long in time consumption, cannot rapidly report clinic and are easy to delay illness state. In addition, the traditional method has lower detection sensitivity and weak specificity, and is difficult to distinguish in a physiological and biochemical mode especially for strains with similar sources. Although bacteria can be identified rapidly by using the traditional PCR (Polymerase Chain Reaction polymerase chain reaction) method, the traditional PCR method has the disadvantages of expensive equipment, complex steps, high requirements on personnel and difficult large-area popularization.
The Loop-mediated isothermal amplification (LAMP-mediated isothermal amplification) is a novel nucleic acid in-vitro amplification technology under the constant temperature condition, and has the characteristics of isothermicity, rapidness, high specificity and sensitivity, simple and convenient product detection and the like. The technology is invented by Notomi et al in 2000 and is now applied to detection of microorganisms such as viruses and fungi and commercial gene sequencing. The method is characterized in that 2 pairs of specific primers aiming at 6 regions of target DNA (generally not more than 300 bp) are designed aiming at selecting target DNA with good specificity and stability, and a macromolecular strand displacement enzyme (Bst DNA polymerase) is utilized to react for 30-60 min under isothermal conditions (60-70 ℃), so that the in-vitro amplification process of nucleic acid can be completed. The LAMP method can follow the principle of circular strand displacement in isothermal environment, and amplified products are in the form of stepped bands with different sizes and distinct layers on nucleic acid electrophoresis. Aiming at the detection of the high-virulence klebsiella pneumoniae, the traditional PCR method is generally adopted at present, and because the equipment required by the PCR method is expensive, the steps are complex, the requirements on personnel are high, and the large-area popularization is not easy, the method for detecting the high-virulence klebsiella pneumoniae is required to be provided more quickly, simply and at lower cost.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, a first object of the present invention is to provide a detection method for rapidly detecting high virulence klebsiella pneumoniae.
The second aim of the invention is to provide a detection kit for rapidly detecting the high-virulence klebsiella pneumoniae.
To achieve the above object, in a first aspect, a detection method for rapidly detecting klebsiella pneumoniae with high virulence according to an embodiment of the present invention includes the steps of:
extracting strain DNA;
placing the extracted strain DNA and the primer into a reaction solution for LAMP amplification reaction;
the reaction solution comprises a primer, wherein the primer is respectively as follows:
upstream outer primer F3:5'-TGGGGTTATTCTTTCGCT-3';
downstream outer primer B3:5'-TTTCCAAGCTTACTGCAATT-3';
upstream inner primer FIP:5'-CCagCAAAacAGCCTAAATACATTG-TGGGGAGTATCTTTGAGAGG-3';
downstream inner primer BIP:5'-TTGGGATACTGTGCTATTTTTCTCT-GGGAAGATGAGAAATACGAGC-3';
upstream loop primer LF:5'-CGCCTCCGTGATGAGGATG-3';
downstream loop primer LB:5'-GCAGAAAAGGGCTAGCGC-3';
after the LAMP amplification reaction is finished, the reaction solution is detected to judge whether the high-virulence klebsiella pneumoniae exists.
Further, according to an embodiment of the present invention, the method for extracting a strain DNA includes:
the experimental bacteria were inoculated on MH agar plates and cultured overnight at 37℃in an incubator;
scraping an appropriate amount of bacteria from MH agar plates into 500ul of autoclaved double distilled water EP tube, and bathing in boiling water for 10min;
cooling in a refrigerator for 10min;
after centrifugation at 12000g for 10min at 4deg.C, the supernatant was taken.
Further, according to an embodiment of the present invention, the mixed solution of the reaction solution is configured to:
12.5. Mu.L of 2 XHNB LAMP mixture;
2.5. Mu.L of 10 XSLAMP primer mix;
0.8M betaine;
1.0. Mu.L of Bst DNA polymerase;
1. Mu.L of template DNA;
25. Mu.L of sterile water.
Further, according to an embodiment of the present invention, the 2.5 μl 10×lamp primer mix concentration is configured to:
the concentrations of the upstream outer primer F3 and the downstream outer primer B3 are respectively as follows: 0.5uM;
the concentrations of the upstream inner primer FIP and the downstream inner primer BIP are respectively as follows: 8uM;
the concentrations of the upstream loop primer LF and the downstream loop primer LB are respectively as follows: 2uM.
Further, according to one embodiment of the present invention, the reaction conditions of the LAMP amplification reaction are:
placing the mixed solution of the LAMP amplification reaction in a water bath environment;
the reaction temperature is as follows: a gradient between 55 and 85 ℃ per 5 ℃;
the reaction time is as follows: between 40 and 70min, one gradient every 5min.
Further, according to an embodiment of the present invention, the reaction conditions of the LAMP amplification reaction are specifically:
the reaction temperature is as follows: 65 ℃;
the reaction time is as follows: 60min;
the termination reaction temperature is: 85 ℃;
the termination reaction time was: 5min.
Further, according to an embodiment of the present invention, the detecting the reaction result to determine whether the high virulence klebsiella pneumoniae exists includes:
taking a reaction solution, detecting a PCR amplification result by agarose gel electrophoresis, and if a gradient band is positioned and the minimum band is 180bp, indicating that the high-virulence klebsiella pneumoniae exists, otherwise, indicating that the high-virulence klebsiella pneumoniae does not exist;
or, adding SYBR green I into the reaction solution for fluorescence detection, wherein if the reaction solution is green, the existence of the high-virulence klebsiella pneumoniae is indicated, otherwise, the existence of the high-virulence klebsiella pneumoniae is indicated.
On the other hand, the invention also provides a detection kit for rapidly detecting the high-virulence klebsiella pneumoniae, which comprises the following components:
the LAMP amplification reaction solution comprises primers which are respectively as follows:
upstream outer primer F3:5'-TGGGGTTATTCTTTCGCT-3';
downstream outer primer B3:5'-TTTCCAAGCTTACTGCAATT-3';
upstream inner primer FIP:5'-CCagCAAAacAGCCTAAATACATTG-TGGGGAGTATCTTTGAGAGG-3';
downstream inner primer BIP:5'-TTGGGATACTGTGCTATTTTTCTCT-GGGAAGATGAGAAATACGAGC-3';
upstream loop primer LF:5'-CGCCTCCGTGATGAGGATG-3';
downstream loop primer LB:5'-GCAGAAAAGGGCTAGCGC-3'.
Further, according to an embodiment of the present invention, the mixed liquid of the reaction liquid is configured to:
12.5. Mu.L of 2 XHNB LAMP mixture;
2.5. Mu.L of 10 XSLAMP primer mix;
0.8M betaine;
1.0. Mu.L of Bst DNA polymerase;
1. Mu.L of template DNA;
25. Mu.L of sterile water.
Further, according to an embodiment of the present invention, the 2.5 μl 10×lamp primer mix concentration is configured to:
the concentrations of the upstream outer primer F3 and the downstream outer primer B3 are respectively as follows: 0.5uM;
the concentrations of the upstream inner primer FIP and the downstream inner primer BIP are respectively as follows: 8uM;
the concentrations of the upstream loop primer LF and the downstream loop primer LB are respectively as follows: 2uM.
The embodiment of the invention provides a detection method and a kit for rapidly detecting high-virulence klebsiella pneumoniae, and the embodiment of the invention establishes a method for rapidly, simply, conveniently and economically detecting the high-virulence klebsiella pneumoniae in sputum by designing 2 pairs of primers to amplify 6 different regions of the high-virulence klebsiella pneumoniae peg-344 gene. The time for detecting the high virulence klebsiella pneumoniae is shorter, and the result can be obtained within 2.5 hours. In addition, the specific primer designed for the specific gene peg-344 gene of the high-virulence klebsiella pneumoniae can be used for outputting a result within 60min at 65 ℃ through condition optimization, electrophoresis detection is not needed, fluorescent observation is directly carried out, the operation is simple and convenient, the result judgment is simple, and the detection cost is reduced. The minimum detection limit of the high virulence klebsiella pneumoniae LAMP reaction system on the genome is 0.475 pg/mu L, the sensitivity is 100 times higher than that of the traditional PCR method, the detection sensitivity is improved, and the specificity is better.
In addition, the establishment of the method provided by the embodiment of the invention can be used for rapidly detecting the high-virulence klebsiella pneumoniae, so that the technology is worthy of further popularization for basic medical institutions, can also be used as a rapid preliminary screening test for monitoring the environment of the food industry and the food safety by basic epidemic prevention departments, and has important significance in epidemiology.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a detection method for rapidly detecting Klebsiella pneumoniae with high virulence provided by an embodiment of the invention;
FIG. 2 is a flowchart of a method for extracting strain DNA according to an embodiment of the present invention;
FIG. 3 is a flowchart of another method for rapidly detecting Klebsiella pneumoniae with high virulence according to an embodiment of the present invention;
FIG. 4 is a flowchart of a detection method for rapidly detecting Klebsiella pneumoniae with high virulence according to an embodiment of the present invention;
FIG. 5 shows the detection results after 2% agarose gel electrophoresis after LAMP amplification reaction according to the embodiment of the present invention;
FIG. 6 is a graph showing the results of a sensitivity test of the LAMP method according to the embodiment of the present invention;
FIG. 7 is a graph showing the experimental result of gel electrophoresis of the detection of DNA loop-mediated isothermal amplification products of a Klebsiella pneumoniae template with ordinary non-high virulence by the LAMP method provided by the embodiment of the invention;
FIG. 8 shows the results of gel electrophoresis of DNA loop-mediated isothermal amplification products of Klebsiella pneumoniae templates for detecting strains of different sources by the LAMP method provided by the embodiment of the invention;
the achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The specific conditions not noted in the examples below are experimental methods, generally according to conditions in conventional conditions, or according to manufacturer's recommendations, and the strains involved in the examples are of the prior art and readily available from published commercial sources by those skilled in the art.
In one aspect, the present invention provides a method for rapidly detecting klebsiella pneumoniae with high virulence, comprising the steps of: s101, extracting strain DNA.
S102, placing the extracted strain DNA and the primers in a reaction solution for LAMP amplification reaction.
Upstream outer primer F3 (5 '. Fwdarw.3'): TGGGGTTATTCTTTCGCT.
Downstream outer primer B3 (5 '. Fwdarw.3'): TTTCCAAGCTTACTGCAATT.
An upstream inner primer FIP (5 '. Fwdarw.3'): CCagCAAAacAGCCTAAATACATTG-TGGGGAGTATCTTTGAGAGG.
Downstream inner primer BIP (5 '. Fwdarw.3'): TTGGGATACTGTGCTATTTTTCTCT-GGGAAGATGAGAAATACGAGC.
Upstream loop primer LF (5 '. Fwdarw.3'): CGCCTCCGTGATGAGGATG.
Downstream loop primer LB (5 '. Fwdarw.3'): GCAGAAAAGGGCTAGCGC.
S103, after the LAMP amplification reaction is finished, detecting the reaction liquid to judge whether high-virulence klebsiella pneumoniae exists; the reaction solution comprises a primer, wherein the primer is respectively as follows:
specifically, in step S101, strain DNA is extracted by thermal pyrolysis, and in the embodiment of the present invention, specific gene peg-344 of klebsiella pneumoniae with high virulence is selected as a target gene and a primer set is designed. And through step S102, placing the strain DNA and the primer group into a LAMP reaction system for LAMP (Loop-mediated isothermal amplification Loop-mediated isothermal amplification technology) amplification reaction. After the diffusion reaction, the result of the diffusion reaction is observed in step S103 to determine whether the extracted strain DNA contains the presence of the highly virulent klebsiella pneumoniae. Because the specific primer in the embodiment is designed for the high-virulence klebsiella pneumoniae, the acid in-vitro amplification of the high-virulence klebsiella pneumoniae sclerotium is completed under the action of the primer and the reaction liquid. LAMP can follow the principle of circular strand displacement in isothermal environment, the amplified products are in the form of stepped strips with different sizes and distinct layers on nucleic acid electrophoresis, and if the reaction solution has stepped strips, the existence of high-virulence klebsiella pneumoniae contained in the extracted strain DNA can be judged. Otherwise, it can be judged that no high virulence klebsiella pneumoniae exists.
The embodiment of the invention designs 2 pairs of primers to amplify 6 different areas of the high-virulence klebsiella pneumoniae peg-344 gene, and establishes a method for rapidly, simply and economically detecting the high-virulence klebsiella pneumoniae in sputum. The time for detecting the high virulence klebsiella pneumoniae is shorter, and the result can be obtained within 2.5 hours. In addition, the embodiment of the invention designs a specific primer aiming at the specific gene peg-344 gene of the high-virulence klebsiella pneumoniae, can output a result within 60min at 65 ℃ through condition optimization, directly can be observed through fluorescence without electrophoresis detection, has simple and convenient operation and simple result judgment, and reduces the detection cost. The minimum detection limit of the high virulence klebsiella pneumoniae LAMP reaction system on the genome is 0.475 pg/mu L, and the sensitivity is 100 times higher than that of the traditional PCR method, thus improving the detection sensitivity. And has better specificity characteristics.
In addition, the establishment of the method provided by the embodiment of the invention can be used for rapidly detecting the high-virulence klebsiella pneumoniae, so that the technology is worthy of further popularization for basic medical institutions, can also be used as a rapid preliminary screening test for monitoring the environment of the food industry and the food safety by basic epidemic prevention departments, and has important significance in epidemiology.
Further, in one embodiment of the present invention, the method for extracting a strain DNA comprises the steps of: s201, inoculating experimental bacteria on MH agar plates, and culturing overnight at 37 ℃ in an incubator.
S202, scraping a proper amount of bacteria from an MH agar plate into an EP tube of 500ul of autoclaved double distilled water, and bathing in boiling water for 10min.
S203, cooling in a refrigerator for 10min.
S204, centrifuging 12000g for 10min at the temperature of 4 ℃ and taking supernatant.
Specifically, the preparation of the PCR template strain DNA can be completed through steps S201 to S204, and the whole operation process is aseptic operation.
Further, in a preferred embodiment of the present invention, the mixed solution of the reaction solution is configured to:
12.5. Mu.L of 2 XHNB LAMP mixture.
2.5. Mu.L of 10 XSLAMP primer mix.
0.8M betaine.
1.0. Mu.L of Bst DNA polymerase.
1. Mu.L of template DNA.
25. Mu.L of sterile water.
The LAMP reaction meets the preferable reaction conditions through the mixed liquid configuration of the reaction liquid.
Further, in a preferred embodiment of the present invention, the 2.5. Mu.L 10×LAMP primer mixture concentration is configured to:
the concentrations of the upstream outer primer F3 and the downstream outer primer B3 are respectively as follows: 0.5uM.
The concentrations of the upstream inner primer FIP and the downstream inner primer BIP are respectively as follows: 8uM.
The concentrations of the upstream loop primer LF and the downstream loop primer LB are respectively as follows: 2uM.
The LAMP reaction meets the preferable reaction conditions through the mixed liquid configuration of the reaction liquid.
Further, in a preferred embodiment of the present invention, the reaction conditions for the LAMP amplification reaction are:
placing the mixed solution of the LAMP amplification reaction in a water bath environment;
the reaction temperature is as follows: a gradient between 55 and 85 ℃ per 5 ℃;
the reaction time is as follows: between 40 and 70min, one gradient every 5min. The LAMP reaction meets the better reaction conditions through the reaction temperature and time conditions of the mixed solution of the reaction solution.
Further, in a preferred embodiment of the present invention, the reaction conditions of the LAMP amplification reaction are specifically:
the reaction temperature is as follows: 65 ℃.
The reaction time is as follows: and 60min.
The termination reaction temperature is: 85 ℃.
The termination reaction time was: 5min.
The LAMP reaction meets the better reaction conditions through the reaction temperature and time conditions of the mixed solution of the reaction solution.
The step of detecting the reaction result to judge whether the high virulence klebsiella pneumoniae exists comprises the following steps:
s303, taking a reaction solution, and detecting a PCR amplification result by agarose gel electrophoresis, wherein if a gradient band is positioned and the minimum band is 180bp, the existence of high-virulence klebsiella pneumoniae is indicated, otherwise, the existence of no high-virulence klebsiella pneumoniae is indicated; or, adding SYBR green I into the reaction solution for fluorescence detection, wherein if the reaction solution is green, the existence of the high-virulence klebsiella pneumoniae is indicated, otherwise, the existence of the high-virulence klebsiella pneumoniae is indicated.
When the reaction solution is taken and the PCR amplification result is detected by agarose gel electrophoresis, the method comprises the following steps:
s403, mixing the reaction solution with a 10×loading buffer, adding the mixture into a sample Loading hole of agarose gel, carrying out 120V electrophoresis for 20min, taking out gel blocks, and obtaining a PCR amplification result through an electrophoresis strip in an ultraviolet imaging system; if the gradient band is located and the minimum band is 180bp, the existence of the high-virulence klebsiella pneumoniae is indicated, otherwise, the existence of the high-virulence klebsiella pneumoniae is indicated.
Further, in one embodiment of the present invention, the stock solution is taken as follows: 0.5uL.
The 10×loading buffer is: 4.5uL.
The reaction solution was mixed with 10×loading buffer and then applied to the Loading well of 2% agarose gel.
Specifically, in steps S203 and S303, after the LAMP reaction was completed, (1) 0.5. Mu.L of the reaction solution was sucked up by a gun head, mixed with 4.5. Mu.L of 10×loading buffer, added to the Loading well of 2% agarose gel, subjected to 120V electrophoresis for 20min, the gel block was taken out, and the electrophoresis band was observed in an ultraviolet imaging system, and photographed for recording. Specific criteria for determining whether a target bacterium is present in a sample are: and detecting the PCR amplification result by agarose gel electrophoresis, wherein if a gradient band is positioned and the minimum band is 180bp, the existence of the high-virulence klebsiella pneumoniae is indicated.
(2) 1 mu L SYBR green I dye is added into the reaction liquid, and the specific standard for determining whether target bacteria exist in the sample is as follows: if the reaction liquid is green, the existence of high-virulence klebsiella pneumoniae is indicated.
Referring to FIG. 5, a comparison of LAMP amplification results is shown, where P is the positive control and N is the negative control. (A) Under the natural illumination condition, after SYBR green I is added, P is green, and N is light yellow. (B) Under the condition of ultraviolet light, after SYBR green I is added, P is bright green, and N is light yellow. (C) (FIG. 5) after electrophoresis on a 2% agarose gel, P appeared as a stepwise band, and N was free of any band. As can be seen from the comparison between FIG. 5 and FIG. 5, the method for rapidly detecting Klebsiella pneumoniae with high virulence provided by the embodiment of the invention can rapidly detect and judge Klebsiella pneumoniae with high virulence.
On the other hand, the invention also provides a detection kit for rapidly detecting the high-virulence klebsiella pneumoniae, which comprises the following components: the LAMP amplification reaction solution comprises primers which are respectively as follows:
upstream outer primer F3 (5 '. Fwdarw.3'): TGGGGTTATTCTTTCGCT.
Downstream outer primer B3 (5 '. Fwdarw.3'): TTTCCAAGCTTACTGCAATT.
An upstream inner primer FIP (5 '. Fwdarw.3'):
CCagCAAAacAGCCTAAATACATTG-TGGGGAGTATCTTTGAGAGG。
downstream inner primer BIP (5 '. Fwdarw.3'):
TTGGGATACTGTGCTATTTTTCTCT-GGGAAGATGAGAAATACGAGC。
upstream loop primer LF (5 '. Fwdarw.3'): CGCCTCCGTGATGAGGATG.
Downstream loop primer LB (5 '. Fwdarw.3'): GCAGAAAAGGGCTAGCGC.
According to the embodiment of the invention, 6 different areas of the high-virulence klebsiella pneumoniae peg-344 gene are amplified by designing 2 pairs of primers, and a kit for rapidly, simply, conveniently and economically detecting the high-virulence klebsiella pneumoniae in sputum is established. The time for detecting the high virulence klebsiella pneumoniae is shorter, and the result can be obtained within 2.5 hours. In addition, the embodiment of the invention designs a specific primer aiming at the specific gene peg-344 gene of the high-virulence klebsiella pneumoniae, can output a result within 60min at 65 ℃ through condition optimization, directly can be observed through fluorescence without electrophoresis detection, has simple and convenient operation and simple result judgment, and reduces the detection cost. The minimum detection limit of the high virulence klebsiella pneumoniae LAMP reaction system on the genome is 0.475 pg/mu L, the sensitivity is 100 times higher than that of the traditional PCR method, the detection sensitivity is improved, and the specificity is better.
In addition, the kit can be established to rapidly detect the high-virulence klebsiella pneumoniae, so that the technology is worthy of further popularization for basic medical units, can be used as a rapid preliminary screening test for monitoring the environment of the food industry and the food safety by basic epidemic prevention departments, and has important significance in epidemiology.
Further, in one embodiment of the present invention, the mixture of the reaction liquids is configured to:
12.5. Mu.L of 2 XHNB LAMP mixture.
2.5. Mu.L of 10 XSLAMP primer mix.
0.8M betaine.
1.0. Mu.L of Bst DNA polymerase.
1. Mu.L of template DNA.
25. Mu.L of sterile water.
The LAMP reaction meets the preferable reaction conditions through the mixed liquid configuration of the reaction liquid.
Further, in one embodiment of the present invention, the 2.5 μl 10×lamp primer mix concentration is configured to:
the concentrations of the upstream outer primer F3 and the downstream outer primer B3 are respectively as follows: 0.5uM.
The concentrations of the upstream inner primer FIP and the downstream inner primer BIP are respectively as follows: 8uM.
The concentrations of the upstream loop primer LF and the downstream loop primer LB are respectively as follows: 2uM.
The LAMP reaction meets the preferable reaction conditions through the mixed liquid configuration of the reaction liquid.
The sensitivity test result of the LAMP method provided by the embodiment of the invention:
referring to FIG. 6, a sensitivity test result diagram of the LAMP method provided by the embodiment of the invention shows that the initial concentration of the genome of high virulence Klebsiella pneumoniae is 475 ng/. Mu.L by using the Take3 program of a microplate spectrophotometer. The template DNA was subjected to concentration gradient dilution, and then isothermal amplification was performed using the original concentrations, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8 dilutions as templates, respectively, and the reaction results were observed by electrophoresis. Compared with the detection sensitivity of the LAMP method and the conventional PCR method, the minimum detection limit of the PCR method is 47.5 pg/mu L, the minimum detection limit of the LAMP method is 0.475 pg/mu L (figure 6), and the sensitivity of the LAMP method established by the test for detecting the Klebsiella pneumoniae is higher.
Wherein the meaning of each letter or number in fig. 6 is:
m is: DL2000 DNA Maker.
N is: negative control.
1 to 9 are reaction solutions having DNA concentrations of 475, 47.5, 4.75, 4.75X10-1, 4.75X10-2, 4.75X10-3, 4.75X10-4, 4.75X10-5 and 4.75X10-6 ng/. Mu.L, respectively.
The embodiment of the invention provides a specific test result of the LAMP method:
referring to FIG. 7 and Table 1, different types of standard strains were inoculated on MH plates, and after overnight incubation in an incubator, DNA template solutions were prepared by picking up, and the detection results showed that none of the other bacteria except for Klebsiella pneumoniae with high virulence could be amplified, as shown in FIG. 7 and Table 1. Tests prove that the detection method has good specificity and good detection effect when being implemented for detection.
As shown in FIG. 7, after the amplification of each strain of Klebsiella pneumoniae which is not a high virulence, the products thereof are subjected to gel electrophoresis, and specifically amplified product bands are visible. Wherein 1 is a positive control; strains 2-16 correspond to Table 1, respectively.
In addition, the amplified products of each strain of common non-high virulent Klebsiella pneumoniae can be visually observed to show transparent dark orange. Wherein 1 positive control; strains 2 to 16 correspond to Table 1 respectively
TABLE 1 specificity test of LAMP method
The LAMP method provided by the embodiment of the invention detects the clinical specimens:
referring to FIG. 8 and Table 2, the test strains were LAMP amplified with 15 high virulence Klebsiella pneumoniae genomic DNAs of AY9630, AY9293, AY8972, AY4992, AY4970, AY2075, AY11489, AP2841, JDZK01, GZK01, GZK02, GZK03, GZK20, XY1298, AP15127 and the like, respectively, and the results were observed and detected by agarose gel electrophoresis (FIG. 8) and direct visual observation, respectively. The results show that both methods can see specific amplification reactions relative to the negative control. It is shown that the primer group can generate positive results with Klebsiella pneumoniae templates with different sources and no false negative results are found.
Wherein, as shown in FIG. 8, after each strain of high virulent Klebsiella pneumoniae is amplified, the products thereof are subjected to gel electrophoresis, and specifically amplified product bands are visible. l, AY9630;2, AY9293;3, AY8972;4, AY4992;5, AY4970;6, AY2075;7, AY11489;8, ap2841;9, jdzk01;10, GZK01;11, GZK02;12, gzk03;13, GZK20;14, XY1298;15, ap15127.
In addition, the amplified products of each strain of high virulent Klebsiella pneumoniae are seen to be characteristic bright green compared with a negative control by human eyes.
TABLE 2 detection results of clinical strains by LAMP method
The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention.
Claims (10)
1. A detection method for rapidly detecting high virulence klebsiella pneumoniae, which is characterized in that the detection method is a diagnosis and treatment method for non-diseases, and comprises the following steps:
extracting strain DNA;
placing the extracted strain DNA and the primer into a reaction solution for LAMP amplification reaction;
the reaction solution comprises a primer, wherein the primer is respectively as follows:
upstream outer primer F3:5'-TGGGGTTATTCTTTCGCT-3';
downstream outer primer B3:5'-TTTCCAAGCTTACTGCAATT-3';
upstream inner primer FIP:
5’-CCagCAAAacAGCCTAAATACATTG-TGGGGAGTATCTTTGAGAGG-3’；
downstream inner primer BIP:
5’-TTGGGATACTGTGCTATTTTTCTCT-GGGAAGATGAGAAATACGAGC-3’；
upstream loop primer LF:5'-CGCCTCCGTGATGAGGATG-3';
downstream loop primer LB:5'-GCAGAAAAGGGCTAGCGC-3';
after the LAMP amplification reaction is finished, the reaction solution is detected to judge whether the high-virulence klebsiella pneumoniae exists.
2. The method for rapidly detecting Klebsiella pneumoniae according to claim 1, wherein the method for extracting strain DNA comprises:
the experimental bacteria were inoculated on MH agar plates and cultured overnight at 37℃in an incubator;
scraping an appropriate amount of bacteria from MH agar plates into 500ul of autoclaved double distilled water EP tube, and bathing in boiling water for 10min;
cooling in a refrigerator for 10min;
after centrifugation at 12000g for 10min at 4deg.C, the supernatant was taken.
3. The method for rapidly detecting klebsiella pneumoniae according to claim 1, wherein the mixture of the reaction solutions is configured to:
12.5 mu L of 2 XHNB LAMP mixed liquor;
2.5. Mu.L of 10×LAMP primer mix;
0.8M betaine;
1.0 [ mu ] L of Bst DNA polymerase;
1 [ mu ] L template DNA;
25 [ mu ] L of sterile water.
4. The method for rapidly detecting klebsiella pneumoniae according to claim 3, wherein the concentration of the 2.5 μl 10×lamp primer mixture is configured as follows:
the concentrations of the upstream outer primer F3 and the downstream outer primer B3 are respectively as follows: 0.5uM;
the concentrations of the upstream inner primer FIP and the downstream inner primer BIP are respectively as follows: 8uM;
the concentrations of the upstream loop primer LF and the downstream loop primer LB are respectively as follows: 2uM.
5. The method for rapid detection of Klebsiella pneumoniae according to claim 4, wherein the reaction conditions of the LAMP amplification reaction are:
placing the mixed solution of the LAMP amplification reaction in a water bath environment;
the reaction temperature is as follows: a gradient between 55 ℃ and 85 ℃ at every 5 ℃;
the reaction time is as follows: between 40 and 70 minutes, one gradient is provided every 5 minutes.
6. The method for rapid detection of Klebsiella pneumoniae according to claim 5, wherein the reaction conditions of the LAMP amplification reaction are as follows:
the reaction temperature is as follows: 65 ℃;
the reaction time is as follows: 60min;
the termination reaction temperature is: 85 ℃;
the termination reaction time was: 5min.
7. The method according to any one of claims 1 to 6, wherein detecting the reaction result to determine whether the high virulent klebsiella pneumoniae exists comprises:
taking a reaction solution, detecting an amplification result by agarose gel electrophoresis, and if a gradient strip is positioned and the minimum strip is 180bp, indicating that high-virulence klebsiella pneumoniae exists, otherwise, indicating that no high-virulence klebsiella pneumoniae exists;
or, adding SYBR green I into the reaction solution for fluorescence detection, wherein if the reaction solution is green, the existence of the high-virulence klebsiella pneumoniae is indicated, otherwise, the existence of the high-virulence klebsiella pneumoniae is indicated.
8. A detection kit for rapidly detecting high virulence klebsiella pneumoniae, comprising:
the LAMP amplification reaction solution comprises primers which are respectively as follows:
upstream outer primer F3:5'-TGGGGTTATTCTTTCGCT-3';
downstream outer primer B3:5'-TTTCCAAGCTTACTGCAATT-3';
upstream inner primer FIP:
5’-CCagCAAAacAGCCTAAATACATTG-TGGGGAGTATCTTTGAGAGG-3’；
downstream inner primer BIP:
5’-TTGGGATACTGTGCTATTTTTCTCT-GGGAAGATGAGAAATACGAGC-3’；
upstream loop primer LF:5'-CGCCTCCGTGATGAGGATG-3';
downstream loop primer LB:5'-GCAGAAAAGGGCTAGCGC-3'.
9. The kit for rapid detection of klebsiella pneumoniae of claim 8, wherein the mixture of reaction solutions is configured to:
12.5 mu L of 2 XHNB LAMP mixed liquor;
2.5. Mu.L of 10×LAMP primer mix;
0.8M betaine;
1.0 [ mu ] L of Bst DNA polymerase;
1 [ mu ] L template DNA;
25 [ mu ] L of sterile water.
10. The detection kit for rapidly detecting high virulence klebsiella pneumoniae according to claim 9, wherein the concentration of the 2.5 μl 10×lamp primer mixture is configured to:
the concentrations of the upstream outer primer F3 and the downstream outer primer B3 are respectively as follows: 0.5uM;
the concentrations of the upstream inner primer FIP and the downstream inner primer BIP are respectively as follows: 8uM;
the concentrations of the upstream loop primer LF and the downstream loop primer LB are respectively as follows: 2uM.
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