CN108279310A - A kind of detection kit and its detection method of Klebsiella Pneumoniae - Google Patents

A kind of detection kit and its detection method of Klebsiella Pneumoniae Download PDF

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CN108279310A
CN108279310A CN201810091934.9A CN201810091934A CN108279310A CN 108279310 A CN108279310 A CN 108279310A CN 201810091934 A CN201810091934 A CN 201810091934A CN 108279310 A CN108279310 A CN 108279310A
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klebsiella pneumoniae
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李梅秀
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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Abstract

The present invention provides a kind of detection kit and its detection method of Klebsiella Pneumoniae.The kit includes that amino acid sequence is 6 polypeptides shown in SEQ ID NO.1~SEQ ID NO.6.The detection method includes step：(1) sample to be tested is detached with separating liquid, the cell of separator well is resuspended in serum free medium；(2) it is that 6 polypeptides shown in SEQ ID NO.1~SEQ ID NO.6 carry out total incubation that amino acid sequence is added into the cell after resuspension, and high specific mouse anti-human IgG antibodies are then added into the system of total incubation carries out antigen-antibody reaction；(3) system after antigen-antibody reaction is washed, adds chromogenic substrate and carries out chromogenic reaction, the spot number that quantal response generates.The kit of the present invention can manually complete sequence synthesize, convenient for production quick, and stability and specificity are high, and kit is easy to operate, significantly reduces cost.

Description

A kind of detection kit and its detection method of Klebsiella Pneumoniae
Technical field
The invention belongs to medical detection technologies, and in particular to a kind of detection kit of Klebsiella Pneumoniae and its inspection Survey method.
Background technology
Klebsiella Pneumoniae is a kind of bacterium (being commonly called as pneumobacillus) mostly important in enterobacteriaceae Klebsiella, Associated diseases account for 95% or more of Klebsiella infection.Klebsiella Pneumoniae is gram negative bacilli, diffusate in lesion It is sticky and weigh, cause interlobar fissure to drop.Klebsiella is strong to extraneous resistance, and drug resistance is also easy to produce to most antibiotic. It can be separated to Klebsiella in the respiratory tract and enteron aisle normal flora of Healthy People, in nature water and cereal.
Klebsiella Pneumoniae is to be clinically separated and one of the important pathogenic bacteria of nosocomial infection, with beta-lactam and amino The broad-spectrum antibiotic such as glycoside are widely used, and bacterium is also easy to produce extended spectrumβ-lactamase (ESBLs) and cephalosporinase (AmpC enzymes) and Aminoglycosides modifying enzyme (AMEs), to common drug include third generation cephalosporin and aminoglycoside is in Reveal serious multi-drug resistant.Hospital infection rate caused by Klebsiella Pneumoniae increases year by year in the recent period, and Multidrug resistance bacterial strain Be continuously increased the failure for often resulting in clinical antibacterial drug therapy and protracted course.Klebsiella pneumoniae Resistance mechanism includes mainly Generate the missing of beta-lactamase, the formation of biofilm, Outer membrane protein.Outer row of antibacterials active etc., antibacterials are resistance to Medicine gene level send out be multidrug resistance bacterial strain clinic aggravation major reason.
The method of detection Klebsiella pneumoniae infection includes mainly at present：Culture of microorganism and Physiology and biochemistry detection, group Knit pathological observation, sediments microscope inspection and PCR, real-time fluorescence PCR equimolecular diagnostic method.Culture of microorganism takes and trains Complicated condition is supported, the interpretation of Physiology and biochemistry testing result depends on the subjective judgement of operator, so as to cause result poor repeatability, Easily misjudgement；Pathological study relies on microtomy, takes；Microscopy relies on smear technique；Molecular Detection such as PCR, It is high that real-time fluorescent PCR technology needs expensive instrument and equipment, technology to require.Klebsiella Pneumoniae can pass through antigen and antibody Specific binding can determine whether pneumonia infection Cray primary according to the quantity of the antibody of Klebsiella Pneumoniae in cycle peripheral blood Bacterium is used for the clinical assistant diagnosis of Klebsiella pneumoniae infection, can quick, sensitivity the infection conditions for detecting human body.Urgently It needs a kind of more convenient effectively specifically for the detection means of Klebsiella Pneumoniae.
Invention content
In view of this, the purpose of the present invention is to provide a kind of detection kit of Klebsiella Pneumoniae and its detection sides Method, to solve above-mentioned at least partly technical problem existing in the prior art.
To achieve the goals above, the present invention provides a kind of detection kit of Klebsiella Pneumoniae comprising amino acid Sequence is 6 polypeptides shown in SEQ ID NO.1~SEQ ID NO.6, respectively：
SEQ ID NO.1：Glu Glu Gly Ala Glu Ala Ala Ala Gly Ile Val Leu Leu Ala Ala Gln Gly Ser Thr Ala；
SEQ ID NO.2：Gln Glu Leu Gly Gly Ile Arg Glu Ser Glu Ala Val Thr Ala Ser Ala Arg Thr Gly Gly；
SEQ ID NO.3：Thr Met Gly Ala Arg Arg Glu Gly Ile Glu Thr Ala Ala Pro Ala Thr Ala Thr Gly Ala；
SEQ ID NO.4：Glu Ala Met Asp Leu Ala Glu Ala Thr Ile Ala Ser Thr Leu Ser Glu Gly Gly Ala Ala；
SEQ ID NO.5：Gly Leu Thr Ile Val Gly Ala Gly Glu Gly Ser Leu Lys Ala Ala Thr Ala Ile Gly Glu；
SEQ ID NO.6：Glu Gly Asp Thr Ala Ile Ser Ala Thr Gly Leu Glu Ala Ala Met Ser Ala Ile Gly Ala。
In the present invention, amino acid sequence is 6 polypeptides shown in SEQ ID NO.1~SEQ ID NO.6 in kit As TB stimulants, for detecting reaction.
Preferably, in the detection kit of the Klebsiella Pneumoniae, amino acid sequence is SEQ ID NO.1~SEQ The content of each in 6 polypeptides shown in ID NO.6 is 10mg.
Preferably, the detection kit of the Klebsiella Pneumoniae further includes serum free medium.
Preferably, the detection kit of the Klebsiella Pneumoniae further includes separating liquid.
Preferably, the detection kit of the Klebsiella Pneumoniae further includes high specific mouse anti-human IgG antibodies and/or lung Scorching Klebsiella infection positive stimulus object.
Preferably, the detection kit of the Klebsiella Pneumoniae further includes the colour developing examination for enzyme-linked immunospot assay Agent, such as NBT/BCIP Color Appearance Systems, final develop the color bluish violet spot or AEC Color Appearance Systems, finally develop the color punctation.
Klebsiella Pneumoniae is detected the present invention also provides the detection kit using aforementioned Klebsiella Pneumoniae Method includes the following steps：
(1) sample to be tested is detached with separating liquid, the cell of separator well is resuspended in serum free medium；
(2) amino acid sequence is added into the cell after resuspension and such as carries out total incubation, then adds into the system of total incubation Enter high specific mouse anti-human IgG antibodies and carries out antigen-antibody reaction；
(3) system after antigen-antibody reaction is washed, adds chromogenic substrate and carries out chromogenic reaction, quantal response The spot number of generation.
In step (1), it is described the cell of separator well is resuspended in serum free medium preferably remain cell concentration be 1.0 ×105~3.0 × 105Cell/mL.
In step (2), the cell after the resuspension is preferably added in 96 orifice plates, and the addition amino acid sequence is SEQ ID 6 polypeptides shown in NO.1~SEQ ID NO.6 are preferably added in 96 orifice plates with the mixing with cells after resuspension to carry out total incubation. The more preferably each reacting hole of the additive amount of cell after the resuspension is 100 μ L, and the amino acid sequence is SEQ ID NO.1 Final concentration of 5 μ L in the preferably each reacting hole of the additive amount of 6 polypeptides shown in~SEQ ID NO.6.It is described to be altogether incubated Preferably 16~20 hours time.
Such as this field routine, step (2) in operation, is preferably provided with positive control and negative control, the positive control For Klebsiella Pneumoniae positive stimulus object；The negative control is left white for serum free medium, i.e., is only added without blood in reacting hole Clear culture medium does not add any other reagent.
It has to be noted that of the present invention belong to for external point the method that Klebsiella Pneumoniae is detected Identify qualitative detection method from sample, only whether to obtain in sample to be tested by Klebsiella Pneumoniae stimulates and infects Quantity is direct result and purpose, is not related to the judgement to human health status, is not belonging to and the relevant method of medical diagnosis on disease.
In the present invention, above-mentioned optimum condition can be combined arbitrarily on the basis of common knowledge of the art to get of the invention each Preferred embodiments.
For the present invention in addition to special instruction, percentage used is all percent by volume.
Beneficial effects of the present invention are：It is provided by the invention include amino acid sequence be SEQ ID NO.1~SEQ ID The detection kit of 6 polypeptides shown in NO.6 can manually complete sequence synthesize, convenient for production quick, stability and specificity Height, and greatly simplified compared to the process of traditional technique in measuring, significantly reduce cost.Using the kit to the external of separation Sample is detected convenient and efficient, and controllability and accuracy are ideal, and kit is easy to operate.
Specific implementation mode
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this hair It is bright to be described in further detail.
The present invention is based on the immune response mechanism of Klebsiella Pneumoniae to be innovated to traditional detection method, develops It is capable of 6 polypeptides of artificial complete sequence synthesis as detectable substance (1~polypeptide of polypeptide 6 i.e. in sequence table, amino acid encoding sequence Row correspond to SEQ ID NO.1~SEQ ID NO.6), by a large amount of experimental verification, and pass through and existing detection method pair Than having a clear superiority, of the invention includes that amino acid sequence is 6 polypeptides shown in SEQ ID NO.1~SEQ ID NO.6 Kit and external Klebsiella pneumoniae infection sample to be tested is detected using it method have it is convenient and efficient, accurate The advantages such as high, at low cost are spent, are had a good application prospect.
Amino acid sequence is 6 polypeptides of SEQ ID NO.1~SEQ ID NO.6, specific to be respectively：
SEQ ID NO.1：Glu Glu Gly Ala Glu Ala Ala Ala Gly Ile Val Leu Leu Ala Ala Gln Gly Ser Thr Ala；
SEQ ID NO.2：Gln Glu Leu Gly Gly Ile Arg Glu Ser Glu Ala Val Thr Ala Ser Ala Arg Thr Gly Gly；
SEQ ID NO.3：Thr Met Gly Ala Arg Arg Glu Gly Ile Glu Thr Ala Ala Pro Ala Thr Ala Thr Gly Ala；
SEQ ID NO.4：Glu Ala Met Asp Leu Ala Glu Ala Thr Ile Ala Ser Thr Leu Ser Glu Gly Gly Ala Ala；
SEQ ID NO.5：Gly Leu Thr Ile Val Gly Ala Gly Glu Gly Ser Leu Lys Ala Ala Thr Ala Ile Gly Glu；
SEQ ID NO.6：Glu Gly Asp Thr Ala Ile Ser Ala Thr Gly Leu Glu Ala Ala Met Ser Ala Ile Gly Ala。
Here is specific embodiment, for the technical solution and technique effect that the present invention is further explained.
Embodiment 1
Human peripheral (PBMC) samples sources in Klebsiella pneumoniae infection patient and non-Klebsiella pneumoniae infection crowd, Quantity is 127 samples to be tested.
Everyone samples and acquires 10mL blood with heparin sodium collecting pipe with individual, is transported to experiment in 6 hours under room temperature Room.
The detecting step of each sample to be tested is as follows：
(1) PBMC is detached with separating liquid, the cell of separator well is resuspended in serum free medium, cell concentration 1.5 ×105Cell/mL；The cell of resuspension is added in PVDF96 orifice plates, 100 μ L are added in each reacting hole, and each sample is added 10 Kong Zhong does 10 repetitions；
(2) be added into each reacting hole respectively reached by serum free medium and final concentration 5 μM of 6 polypeptides form it is mixed 50 μ L of object are closed, is uniformly mixed and carries out total incubation；Positive control and negative control are set, are added into the system of total incubation high special Property mouse anti-human IgG antibodies carry out antigen-antibody reaction；
(3) 6% 36 DEG C of carbon dioxide incubators are incubated 16 hours；
(4) system after antigen-antibody reaction is washed with PBS buffer solution；
(5) it adds chromogenic substrate and carries out chromogenic reaction, be incubated at room temperature 25 minutes, spot formation is observed during being incubated, The spot number that quantal response generates.
Divided according to negative and Positive judgement standards judgement using the quantity of spot-analysis instrument meter number spot formation cell Other statistic mixed-state feminine gender and positive number of cases, as shown in the table.
Positive coincidence rate：74/ (74+5)=93.67%
Negative match-rate：43/ (43+5)=89.58%
Embodiment 2
Human peripheral (PBMC) samples sources in Klebsiella pneumoniae infection patient and non-Klebsiella pneumoniae infection crowd, Quantity is 127 samples to be tested.
Everyone samples and acquires 10mL blood with heparin sodium collecting pipe with individual, is transported to experiment in 6 hours under room temperature Room.
The detecting step of each sample to be tested is as follows：
(1) PBMC is detached with separating liquid, the cell of separator well is resuspended in serum free medium, cell concentration 3.5 ×105Cell/mL；The cell of resuspension is added in PVDF96 orifice plates, 100 μ L are added in each reacting hole, and each sample is added 10 Kong Zhong does 10 repetitions；
(2) be added into each reacting hole respectively reached by serum free medium and final concentration 5 μM of 6 polypeptides form it is mixed 50 μ L of object are closed, is uniformly mixed and carries out total incubation；Positive control and negative control are set, are added into the system of total incubation high special Property mouse anti-human IgG antibodies carry out antigen-antibody reaction；
(3) 5% 36 DEG C of carbon dioxide incubators are incubated 20 hours；
(4) system after antigen-antibody reaction is washed with PBS buffer solution；
(5) it adds chromogenic substrate and carries out chromogenic reaction, be incubated at room temperature 30 minutes, spot formation is observed during being incubated, The spot number that quantal response generates.
Divided according to negative and Positive judgement standards judgement using the quantity of spot-analysis instrument meter number spot formation cell Other statistic mixed-state feminine gender and positive number of cases, as shown in the table.
Positive coincidence rate：77/ (77+3)=96.25%
Negative match-rate：45/ (45+2)=95.74%
There are similar sensitivity and specificity in experimental result surface compared with the existing product using same procedure, It can be used as a kind of Klebsiella Pneumoniae detection kit, and of low cost, it is easy to operate.
In conclusion mixtures of polypeptides, detection kit and its corresponding detection method of the present invention are for detecting pneumonia Klebsiella has the characteristics that convenient and efficient, stability is high, high specificity, and significantly reduces cost.
The above is only presently preferred embodiments of the present invention, is not imposed any restrictions to the present invention, every according to the present invention Technical spirit changes any simple modification, change and equivalent structure made by above example, still falls within skill of the present invention In the protection domain of art scheme.
Sequence table
<110>Li Xiumei
<120>A kind of detection kit and its detection method of Klebsiella Pneumoniae
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Glu Glu Gly Ala Glu Ala Ala Ala Gly Ile Val Leu Leu Ala Ala Gln
1 5 10 15
Gly Ser Thr Ala
20
<210> 2
<211> 20
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
Gln Glu Leu Gly Gly Ile Arg Glu Ser Glu Ala Val Thr Ala Ser Ala
1 5 10 15
Arg Thr Gly Gly
20
<210> 3
<211> 20
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Thr Met Gly Ala Arg Arg Glu Gly Ile Glu Thr Ala Ala Pro Ala Thr
1 5 10 15
Ala Thr Gly Ala
20
<210> 4
<211> 19
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 4
Glu Ala Met Asp Leu Ala Glu Ala Thr Ile Ala Ser Thr Leu Glu Gly
1 5 10 15
Gly Ala Ala
<210> 5
<211> 20
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 5
Gly Leu Thr Ile Val Gly Ala Gly Glu Gly Ser Leu Lys Ala Ala Thr
1 5 10 15
Ala Ile Gly Glu
20
<210> 6
<211> 20
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 6
Glu Gly Asp Thr Ala Ile Ser Ala Thr Gly Leu Glu Ala Ala Met Ser
1 5 10 15
Ala Ile Gly Ala
20

Claims (7)

1. a kind of detection kit of Klebsiella Pneumoniae, which is characterized in that it include amino acid sequence be SEQ ID NO.1~ 6 polypeptides shown in SEQ ID NO.6：
SEQ ID NO.1：Glu Glu Gly Ala Glu Ala Ala Ala Gly Ile Val Leu Leu Ala Ala Gln Gly Ser Thr Ala；
SEQ ID NO.2：Gln Glu Leu Gly Gly Ile Arg Glu Ser Glu Ala Val Thr Ala Ser Ala Arg Thr Gly Gly；
SEQ ID NO.3：Thr Met Gly Ala Arg Arg Glu Gly Ile Glu Thr Ala Ala Pro Ala Thr Ala Thr Gly Ala；
SEQ ID NO.4：Glu Ala Met Asp Leu Ala Glu Ala Thr Ile Ala Ser Thr Leu Ser Glu Gly Gly Ala Ala；
SEQ ID NO.5：Gly Leu Thr Ile Val Gly Ala Gly Glu Gly Ser Leu Lys Ala Ala Thr Ala Ile Gly Glu；
SEQ ID NO.6：Glu Gly Asp Thr Ala Ile Ser Ala Thr Gly Leu Glu Ala Ala Met Ser Ala Ile Gly Ala。
2. the detection kit of Klebsiella Pneumoniae according to claim 1, which is characterized in that in the kerekou pneumonia primary In the detection kit of bacterium, amino acid sequence is each in 6 polypeptides shown in SEQ ID NO.1~SEQ ID NO.6 Content be 10mg.
3. the detection kit of Klebsiella Pneumoniae according to claim 1, which is characterized in that the Klebsiella Pneumoniae Detection kit further include serum free medium.
4. the detection kit of Klebsiella Pneumoniae according to claim 1, which is characterized in that the Klebsiella Pneumoniae Detection kit further include separating liquid.
5. the detection kit of Klebsiella Pneumoniae according to claim 1, which is characterized in that the Klebsiella Pneumoniae Detection kit further include high specific mouse anti-human IgG antibodies and/or Klebsiella pneumoniae infection positive stimulus object.
6. the detection kit of Klebsiella Pneumoniae according to claim 1, which is characterized in that the Klebsiella Pneumoniae Detection kit further include colour reagent for enzyme-linked immunospot assay.
7. using Klebsiella Pneumoniae as described in any one of claim 1-6 detection kit to Klebsiella Pneumoniae into The method of row detection, which is characterized in that include the following steps：
(1) sample to be tested is detached with separating liquid, the cell of separator well is resuspended in serum free medium；
(2) it is 6 polypeptides shown in SEQ ID NO.1~SEQ ID NO.6 that amino acid sequence is added into the cell after resuspension Total incubation is carried out, high specific mouse anti-human IgG antibodies are then added into the system of total incubation carries out antigen-antibody reaction；
(3) system after antigen-antibody reaction is washed, adds chromogenic substrate and carry out chromogenic reaction, quantal response generates Spot number.
CN201810091934.9A 2018-01-30 2018-01-30 A kind of detection kit and its detection method of Klebsiella Pneumoniae Withdrawn CN108279310A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456409A (en) * 2018-11-13 2019-03-12 昆明理工大学 For detecting the specific antibody group and application of klebsiella pneumoniae
CN110540595A (en) * 2018-12-20 2019-12-06 湖北工业大学 preparation method of klebsiella pneumoniae antibody latex microsphere immunochromatography detection test paper
WO2022091531A1 (en) * 2020-10-28 2022-05-05 国立大学法人北海道大学 Anti-tumor peptide and use thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456409A (en) * 2018-11-13 2019-03-12 昆明理工大学 For detecting the specific antibody group and application of klebsiella pneumoniae
CN109456409B (en) * 2018-11-13 2021-10-29 昆明理工大学 Specific antibody group for detecting klebsiella pneumoniae and application thereof
CN110540595A (en) * 2018-12-20 2019-12-06 湖北工业大学 preparation method of klebsiella pneumoniae antibody latex microsphere immunochromatography detection test paper
WO2022091531A1 (en) * 2020-10-28 2022-05-05 国立大学法人北海道大学 Anti-tumor peptide and use thereof

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Application publication date: 20180713