CN109750085A - A kind of detection method precisely quickly detecting target superbacteria - Google Patents
A kind of detection method precisely quickly detecting target superbacteria Download PDFInfo
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Abstract
The application belongs to medical sanitary technology field, and in particular to a kind of detection method for precisely quickly detecting target superbacteria.This method is using flcating germ in environment as sample acquisition target；Specific method includes: sampling and is enriched with, screening and culturing, qualitatively or quantitatively determines evaluation.The characteristic that the application can make luciferase produces chemiluminescence luminous using the ATP that the bacterium that phage splitting is in existing state is discharged, so as to the more intuitive qualitative or quantitative type or content for determining " superbacteria ", and due to the specificity of bacteriophage, even if therefore in containing the sample for mixing bacterium, without being separately cultured, judgement can quickly and be precisely detected, therefore there is preferable application value.
Description
Technical field
The application belongs to medical sanitary technology field, and in particular to a kind of precisely quickly detection of detection target superbacteria
Method.
Background technique
" superbacteria " is the general designation of a kind of bacterium, its main feature is that have powerful drug resistance to most of antibiotic, it can
To propagate in the mankind, animal and environment, increase the treatment difficulty to the infected, treated effect declines, case fatality rate rises,
Medical expense is substantially increased, especially to hypoimmunity, long-time service antibacterials and receiving invasion operation in inpatient
The patients such as treatment have larger threat." global " superbacteria " monitoring report " display of World Health Organization's publication in 2014,
The death rate for infecting " superbacteria " patient is about 2 times for infecting not drug-resistant bacteria patient.And due to antibiotics
It is commonly used and spread unchecked using etc. reasons, lead to the situation of " superbacteria " just in whole world diffusion, therefore " whole world is " super thin
Bacterium " monitoring report " think " if adopted an effective measure not in time, it is dead that common infection will lead to large-scale crowd ".
In medical practice, most commonly seen and particularly important several superbacterias include: Carbapenem-resistant Bao Man not
Lever bacterium (carbapenem-resistant Acinetobacter baumannii, CRAB/CRA), Carbapenem-resistant
Pseudomonas aeruginosa (carbapenem-resistant Pseudomonas aeruginosa, CRPA/CRP), carbapenem
Class drug resistance Klebsiella Pneumoniae (carbapenem-resistant Klebsiella pneumonia, CRKP/CRK), carbon are green
Mould alkenes drug resistance escherichia coli (carbapenem-resistant Escherichia coli, CREC/CRE) etc., Yin Qinai
Medicine situation is the most serious and is easiest to send out prevalence, therefore is classified as particularly important superbacteria, and Ying Yinqi is paid close attention to.
In the medical environments such as hospital, commonly using disinfection and sterilization mode is ultraviolet light and disinfectant, and is most also easy to produce super
The place of grade bacterium.And above-mentioned four kinds of typical super bacteriums are because having a stronger resistivity to disinfectant and ultraviolet light, therefore are also
Cause the common pathogen of inside-hospital infection.They can exist for a long time in hospital environment, and can be colonized in medical staff
Respiratory tract, routine disinfection measure is difficult to thoroughly remove, therefore brings more serious health hidden to patient and medical staff
Suffer from.Also therefore, for these superbacterias it is how fast and accurate screening, determine its concrete type, and in turn determine most
For suitable therapeutic modality, it is to ensure that the important prerequisite of patient and medical staff's health.
Summary of the invention
The application is designed to provide a kind of detection method for precisely quickly detecting target superbacteria, thus to be related super
The immunotherapy targeted autoantibody of grade bacterium establishes technical foundation.
Details are as follows for the technical solution that the application is taken.
It is a kind of that precisely quickly the detection method of detection target superbacteria, this method are acquired by sample of flcating germ in environment
Object, as example (in fact, the method that this patent provides can also be used for any kind of Samples detection, such as body surface, skin
Skin mucous membrane surface, sputum, excrement, fester, blood etc. contain germy sample), mainly to whether containing in flcating germ in environment
Superbacteria carries out screening and identification containing which kind of superbacteria and superbacteria content situation, thus to be further directed to
Property prevention and treatment lay the foundation；
During screening and identification, using LB culture solution as culture medium when flcating germ culture；
Specific selective mechanisms method includes the following steps:
(1) it samples and is enriched with
It acquires in environment after flcating germ, (contains carbapenem antibiotic, as Imipenem or Metro are trained in sterile LB culture solution
South, concentration referring to the drug resistance break in CLSI to screen out non-superbacteria, specific for example, 4 ~ 16ug/ml) in carry out it is preliminary
Enrichment culture；
The acquisition environment is specifically, for example, flcating germ in air environment；
When enrichment culture, for different target superbacteria, different condition of culture can be designed, but super thin for common target
Bacterium is generally incubated condition are as follows: 37 DEG C, 200rpm concussion；
The frequent goal superbacteria, it is specific to be, for example: Carbapenem-resistant Acinetobacter bauamnnii (carbapenem-
Resistant Acinetobacter baumannii, CRAB/CRA), Carbapenem-resistant pseudomonas aeruginosa
(carbapenem-resistant Pseudomonas aeruginosa, CRPA/CRP), Carbapenem-resistant pneumonia gram
The primary bacterium of thunder (carbapenem-resistant Klebsiella pneumonia, CRKP/CRK), Carbapenem-resistant are big
Uncommon bacterium of intestines angstrom (carbapenem-resistant Escherichia coli, CREC/CRE) etc.；
(2) screening and culturing
Test sample after preliminary concentration in step (1) is sampled, portion is used as control sample, and portion is used as screening test sample；
In screening test sample, fluorescein and luciferase is added, while bacteriophage is added to final concentration up to 108PFU/ml with
On, continue to cultivate 0.5h ~ 1h or so so that bacteriophage sufficiently cracks target superbacteria；
In control sample, fluorescein is added and luciferase, similarity condition are cultivated；
It is to be understood that due to having specificity between bacteriophage and target superbacteria, that is, phage specificities act on
Specific objective superbacteria bacterial strain, therefore before for the detection of specific objective superbacteria, it needs to screen determining super for target
The specific bacteriophage of grade bacterium；And it is more convenient realization this purpose, when adding bacteriophage, specific bite can be constructed first
Thallus library utilizes commercialization phage library, so as to more easily realize coherent detection process；
(3) qualitatively or quantitatively determine evaluation
Using luminometer, screening test sample in step (2) and control sample fluorescence intensity are measured and are recorded respectively；From
And whether qualitatively or quantitatively determined containing target superbacteria and superbacteria content in acquisition environment；
When qualitative judgement, after specific bacteriophage is added, if screening test sample occurs being not less than 4 times of fluorescence intensity change situations,
It so can be determined that in acquisition environment and contain the corresponding superbacteria bacterial strain of the bacteriophage；
When quantitative judgement, using the sample time in step (2) as evaluation points, with screening test sample cracking after fluorescence intensity with
The ratio of the fluorescence intensity of control sample evaluates superbacteria content in acquisition environment as evaluation criterion；
The ratio of the fluorescence intensity of fluorescence intensity and control sample is not less than 10 after the screening test sample cracking.
It is to be understood that the main technical principle of the application is: bacteriophage is that one kind being capable of specific infection its host
Bacterium and by rapid cleavage virus, specificity it is very strong, therefore phagocytosis spectrum it is very narrow.Under normal conditions, a kind of bacteriophage is only capable of
One or more of bacteriums are cracked, to other bacteriums without effect, and the bacterium that can be cracked by certain phage specificities is known as this
The host strain of kind bacteriophage.The main technical principle that the application is detected using fluorescence are as follows: ATP(Adenosine
Triphosphate, atriphos) it is a kind of kinetomeres that live bacteria is intracellular, it is the energy object of bacterial metabolism
Matter；And luciferase (Luciferase) is during produces chemiluminescence (Luciferin) oxyluminescence, it is necessary to depend on ATP
Participation just can be carried out, therefore centainly can produce in the ATP for thering is live bacteria to be discharged fluorescence (but phage splitting
The ATP of the bacterium initial stage meeting a large amount of viable bacterias of instantaneous relase, so that making fluorescence intensity that emergentness occur increases variation)；And it is dead
Bacterium (after phage splitting host strain, host strain be death), due to the stopping of metabolism, cell no longer generates ATP, because
This can not issue fluorescence.
Preliminary Applications effect shows screening technique provided herein, has following technical advantage:
(1) it detects high specificity: due to the high degree of specificity of phage splitting bacterium, can realize to target superbacteria
Accurate tracking；
(2) detection speed is fast, high sensitivity: Preliminary Applications experiment shows when that will extend to 6h in the reaction time, to target bacteria
The limting concentration of detection is 1 ~ 10 CFU/ml, shows preferable sensitivity, and the reaction time is more controllable；
(3) operating method is relatively simple: detection screening process in, without to containing mixed cell sample carry out bacterium separation and
The pure culture of target bacteria, can determine the existing state and quantity of target bacteria in mixed microorganism, therefore can effectively drop
Low operation difficulty.
Generally, the application can make luciferase using the ATP that the bacterium that phage splitting is in existing state is discharged
The luminous characteristic of produces chemiluminescence, so as to the more intuitive qualitative or quantitative type or content for determining " superbacteria ", and
Due to the specificity of bacteriophage, even if in containing the sample for mixing bacterium, it, can be quickly and accurate without being separately cultured
Detection determines, therefore has preferable application value.
Detailed description of the invention
Fig. 1 is that different type Acinetobacter bauamnnii bacteriophage is (every to the testing result of corresponding positive sample and ' negative ' specimens
Group left side is ' negative ' specimens, and there is positive sample on right side)；
Fig. 2 is testing result (every group left side of the different type pseudomonas aeruginosa phagocytosis to corresponding positive sample and ' negative ' specimens
For ' negative ' specimens, there is positive sample on right side)；
Fig. 3 is that (every group left for testing result of the different type Klebsiella Pneumoniae bacteriophage to corresponding positive sample and ' negative ' specimens
Side is ' negative ' specimens, and there is positive sample on right side)；
Fig. 4 is that (every group of left side is testing result of the different type coliphage to corresponding positive sample and ' negative ' specimens
There is positive sample on ' negative ' specimens, right side)；
Fig. 5 ~ Figure 10, respectively superbacteria initial concentration are respectively 106At CFU/ml(1h be positive sample), 105 CFU/ml
(being positive sample at 2h), 104At CFU/ml(3h be positive sample), 103 At CFU/ml(4h be positive sample), 102
At CFU/ml(5h be positive sample), 101At CFU/ml(6h be positive sample) examination criteria curve.
Specific embodiment
Explanation is further explained to the application below with reference to embodiment.Before introducing specific embodiment, with regard to following realities
It applies Experimental Background situation in part in example and briefly introduces and be described as follows.
Biomaterial:
It should be noted that relevant bacteria species employed in following embodiments and corresponding bacteriophage are according to a conventional method from yard
Acquire acquisition, for ease of description and distinguish, inventor voluntarily encoded (coding without particular meaning, only convenient for record and
Description), and in particular to strain (and bacterial strain) and corresponding bacteriophage have:
4 plants of Acinetobacter bauamnniis (AB1, AB1, AB2, AB3 and AB09V) and Specific lytic kill the bacteriophage of these bacterial strains
Its corresponding host strain of PAB1, PAB1, PAB2, PAB3, ZZ1(is respectively AB1, AB1, AB2, AB3, AB09V)；
4 Pseudomonas aeruginosa strains (PA1, PA2, PA3 and PA4) and Specific lytic kill these bacterial strains bacteriophage PPA1,
Its corresponding host strain of PPA2, PPA3 and PPA4(is respectively PA1, PA2, PA3 and PA4)；
4 plants of Klebsiella Pneumoniaes (KP1, KP2, KP3 and KP4) and Specific lytic kill these bacterial strains bacteriophage PKP1,
Its corresponding host strain of PKP2, PKP3 and PKP4(is respectively KP1, KP2, KP3 and KP4)；
4 plants of Escherichia coli (EC1, EC2, EC3 and EC4) and Specific lytic kill these bacterial strains bacteriophage PEC1, PEC2,
Its corresponding host strain of PEC3 and PEC4(is respectively EC1, EC2, EC3 and EC4)；
It is identified it should be noted that above-mentioned bacterial strains are sequenced by 16sRNA, confirms that its strain is errorless, and referring to NCCLS medicine
Quick experimental standard verifying, confirms that it is the superbacteria of imipenem-resistant；
It should be noted that being not limited to above-mentioned bacterial strains and corresponding bacteriophage in practical selective mechanisms operation；And due to tool
Body specific environment summarizes superbacteria type and bacterial strain is relatively fixed (excellent under normal circumstances, with season, changes in environmental conditions
The super strain of gesture, bacterial strain can change a lot, but overall situation is relatively stable), therefore to be easy to operate, before selective mechanisms,
Can targetedly be constructed for the microbial strains (including superbacteria) in the presence of environment to be detected phage library (under it is true
It applies example verifying to carry out using the bacteriophage that inventor voluntarily constructs as experiment basis, and is constructed about phage library, with reference to existing
Have technology building, be no longer described in detail), consequently facilitating subsequent operation.
Experiment reagent:
LB culture solution used by during strain culturing is prepared in the usual way, when antibiotic is added, is trained using LB
Carbapenem antibiotic (16 μ g/ml Imipenem) is added before nutrient solution；
Luciferase, fluorescein are Sigma Products, refer to its specification using concentration, application method；
Laboratory apparatus:
TS100 constant temperature blending instrument, the auspicious sincere Products in Hangzhou；
Glomax luminometer, Promega Products.
Embodiment 1
The present embodiment is briefly situated between for determining superbacteria using the qualitative detection of fluorescent emission properties with regard to coherent detection process
It continues as follows.
It should be noted that prepared sample is aggregate sample in experimentation for the specificity for further proving bacteriophage
(that is, being the mixture of a variety of superbacterias in mixing sample).
(1) sample mixing and enrichment culture
Common superbacteria standard sample (in advance respectively screening and identification and save) is inoculated with respectively and (is contained into sterile LB culture solution
The carbapenem antibiotics imipenem of 16ug/ml) in, mixed culture is used as aggregate sample.
In the present embodiment, when mixed culture, general condition of culture are as follows: 37 DEG C, 200rpm shake culture it is dense to superbacteria
Degree is not less than 106 CFU/ml；
Involved superbacteria (specific bacterial strain is referring to preceding description) in the frequent goal superbacteria namely the present embodiment,
It specifically includes:
Carbapenem-resistant Acinetobacter bauamnnii (carbapenem-resistant Acinetobacter baumannii,
CRAB/CRA),
Carbapenem-resistant pseudomonas aeruginosa (carbapenem-resistant Pseudomonas aeruginosa,
CRPA/CRP),
Carbapenem-resistant Klebsiella Pneumoniae (carbapenem-resistant Klebsiella pneumonia,
CRKP/CRK),
Carbapenem-resistant escherichia coli (carbapenem-resistant Escherichia coli, CREC/CRE).
(2) screening and culturing
Aggregate sample in step (1) is sampled, for portion as control sample (fluorescein and luciferase is added), portion is used as test sample
(same that fluorescein and luciferase is added)；
In experimentation, the use reference book of fluorescein and luciferase, fluorescent strength determining is sent out using Glomax
Optical detector detection record；
In test sample, different type bacteriophage in phage library is selected to be separately added into (that is, containing only one in every part of phage library
Kind of bacteriophage, and test sample is divided into more parts and is tested respectively) to bacteriophage final concentration up to 108PFU/ml or more；Then right
Continue shake culture 0.5h with test sample in the same old way, so that bacteriophage sufficiently cracks corresponding superbacteria.
(3) it measures
Using luminometer, test sample in step (2) and control sample fluorescence intensity are measured and are recorded respectively.
Experimental result is as shown in Fig. 1 ~ Fig. 4.Analysis can be seen that in control sample (i.e. negative sample), due to not having phagocytosis
Body cracking, therefore fluorescence intensity never has significant change；And simulate in sample, (Fig. 1 ~ 4 are to add after addition specific type bacteriophage
Enter fluorescence intensity change situation caused by every kind of bacteriophage), bacteriophage is added front and back fluorescence intensity and substantially change, and fluorescence is strong
Degree variation is emphasized minimum also at 4 times or so, and the sensitivity of this numbers illustrated detection method is higher, can be used for qualitatively judging ring
Whether contain target superbacteria in border.In other words, specifically qualitatively judging in environment whether contain specific type superbacteria
When, after being acted on specific phage splitting, whether same sample fluorescence intensity has at least 4 times or more situations of change to carry out qualitative sample
Whether contain corresponding superbacteria in product.
Embodiment 2
The present embodiment corresponds to bacteriophage with the general drug resistance Acinetobacter bauamnnii AB09V(of particular super bacterium as ZZ1) sxemiquantitative inspection
For the building of survey method, coherent detection process is briefly discussed below.
(1) it samples and prepares simulation sample
Using flcating germ air sampler to acquire flcating germ in environment, as background sample (without superbacteria in background sample), early period adds
Entering various concentration, (additional amount is respectively 101、102、103、104、105、106CFU/ml) general drug resistance Acinetobacter bauamnnii AB09V system
The standby simulation sample (reflecting that concentration is added in Acinetobacter bauamnnii AB09V with sample time) for containing superbacteria, and sterile
Preliminary concentration culture is carried out in LB culture solution, 37 DEG C, 200rpm shake culture, when subsequent experimental is sampled every 1h, to examine
Examining different superbacteria concentration in sample influences situation for fluorescence intensity change；
(2) it cultivates
The simulation sample of concentration superbacteria sample each in step (1) is divided into two parts, portion is used as control sample, and portion is as examination
Sample is tested, fluorescein and luciferase are separately added into, then bacteriophage ZZ1 is added to final concentration up to 10 in test sample8PFU/ml, will
Control sample and 37 DEG C again of test sample, 200rpm shake culture 0.5h, so that test sample pnagus medius sufficiently cracks superbacteria.It is real
During testing, control sample, test sample different phase absorbance are recorded, measured and compared respectively.
Different sample time change in fluorescence situations are measured and are drawn.Abscissa is (to train sample time when drawing
The time is supported, reflects superbacteria content situation in simulation sample indirectly by incubation time situation), ordinate is fluorescence intensity intensity
Increase the logarithm of multiple, that is, fluorescence intensity/control sample fluorescence intensity after lg(phage splitting).Fig. 5 ~ 10 are that difference initially contains
Measure the standard curve of fluorescence intensity change of the LB culture solution of target superbacteria (AB09V) after by bacteriophage ZZ1 effect.
Using logarithm >=1 of fluorescence intensity increase multiple, as standard, (>=1, i.e. fluorescence intensity increases at least 10 times, with this
Be as the main reason for evaluation criterion: Glomax luminometer is very high to the measurement sensitivity of fluorescence intensity, not equally
When product, different uciferase activities slightly have difference, detected value is easy to appear certain difference, therefore increased times of Positive fluorescence intensity
Number accepted standard reference values setting it is higher, a possibility that experimental error causes false positive, is just smaller, is based on many experiments feelings
The case where condition, 10 multiple value of comprehensive selection can preferably overcome false positive).
From fig. 5, it can be seen that culture 1h or so can reach this standard, it is based on this incubation time, it can further reverse push
Leading superbacteria content situation in sxemiquantitative original analog sample is about 106CFU/ml；
Similarly, from Fig. 6, it can be seen that culture 2h or so can reach this evaluation criterion, be based on this incubation time, can be into one
Walking superbacteria content situation in reverse-direction derivation sxemiquantitative original analog sample is about 105CFU/ml；
In Fig. 7, it can be seen that culture 3h or so can reach this evaluation criterion, be based on this incubation time, can further reverse push
Leading superbacteria content situation in sxemiquantitative original analog sample is about 104CFU/ml；
In fig. 8, it can be seen that culture 4h or so can reach this evaluation criterion, it is based on this incubation time, it can further reverse push
Leading superbacteria content situation in sxemiquantitative original analog sample is about 103CFU/ml；
In fig. 9, it can be seen that culture 5h or so can reach this evaluation criterion, it is based on this incubation time, it can further reverse push
Leading superbacteria content situation in sxemiquantitative original analog sample is about 102CFU/ml；
In Figure 10, it can be seen that culture 6h or so can reach this evaluation criterion, be based on this incubation time, can be further reversed
Deriving superbacteria content situation in sxemiquantitative original analog sample is about 10CFU/ml.
In other words, increase by 10 times as quantitative assessment criteria using fluorescence intensity, the time by reaching this standard for the first time can
With superbacteria content situation in reverse-direction derivation primary sample, and then can be super in the practical killing environment of bacteriophage further to instruct
The reasonable employment of grade bacterium lays the foundation.
Claims (6)
1. a kind of detection method for precisely quickly detecting target superbacteria, which is characterized in that this method is with super thin in environment
Bacterium is target object；Specific selective mechanisms method includes the following steps:
(1) it samples and is enriched with
It acquires in environment after flcating germ, preliminary concentration culture is carried out in the culture medium containing antibiotic；
The antibiotic is Imipenem or Meropenem；
The application superbacteria, are as follows: Carbapenem-resistant Acinetobacter bauamnnii, Carbapenem-resistant pseudomonas aeruginosa,
Carbapenem-resistant Klebsiella Pneumoniae, Carbapenem-resistant escherichia coli；
(2) screening and culturing
Test sample after preliminary concentration in step (1) is sampled, portion is used as control sample, and portion is used as screening test sample；
In screening test sample, fluorescein and luciferase is added, while bacteriophage is added, continues to cultivate 0.5h ~ 1h so as to bite
Thallus sufficiently cracks target superbacteria；
In control sample, fluorescein is added and luciferase, similarity condition are cultivated；
(3) qualitatively or quantitatively determine evaluation
Using luminometer, screening test sample in step (2) and control sample fluorescence intensity are measured and are recorded respectively；From
And whether qualitatively or quantitatively determined containing target superbacteria and superbacteria content in acquisition environment；
When qualitative judgement, after specific bacteriophage is added, if screening test sample occurs being not less than 4 times of fluorescence intensity change situations,
Determine to contain the corresponding superbacteria bacterial strain of the bacteriophage in acquisition environment；
When quantitative judgement, using the sample time in step (2) as evaluation points, with screening test sample cracking after fluorescence intensity with
The ratio of the fluorescence intensity of control sample evaluates superbacteria content in acquisition environment as evaluation criterion.
2. precisely quickly detecting the detection method of target superbacteria as described in claim 1, which is characterized in that screening and identification mistake
Cheng Zhong, using LB culture solution as culture medium when flcating germ culture.
3. precisely quickly detecting the detection method of target superbacteria as claimed in claim 2, which is characterized in that in step (1),
Drug resistance break of the antibiotic concentration referring to CLSI in LB culture solution.
4. precisely quickly detecting the detection method of target superbacteria as described in claim 1, which is characterized in that in step (2),
Bacteriophage is added final concentration of 108PFU/ml or more.
5. precisely quickly detecting the detection method of target superbacteria as described in claim 1, which is characterized in that in step (1),
Enrichment culture condition are as follows: 37 DEG C, 200rpm shake culture.
6. precisely quickly detecting the detection method of target superbacteria as described in claim 1, which is characterized in that in step (3),
When quantitative judgement, the ratio of the fluorescence intensity of fluorescence intensity and control sample is not less than 10 after the cracking of screening test sample.
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| CN110484595A (en) * | 2019-10-10 | 2019-11-22 | 希拓生物科技有限公司 | A kind of evaluation method being atomized bacteriophage bactericidal effect |
| CN114121246A (en) * | 2021-11-24 | 2022-03-01 | 杭州杏林信息科技有限公司 | Hospital environment cleaning and disinfection management method and system |
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