CN104165879B - For the method identifying Legionella virulence - Google Patents
For the method identifying Legionella virulence Download PDFInfo
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- CN104165879B CN104165879B CN201410409558.5A CN201410409558A CN104165879B CN 104165879 B CN104165879 B CN 104165879B CN 201410409558 A CN201410409558 A CN 201410409558A CN 104165879 B CN104165879 B CN 104165879B
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Abstract
The invention discloses a kind of method for identifying Legionella virulence and dedicated kit thereof.The method of the present invention comprises the steps: that (1) arranges parameter: Spectral acquisition setup；Grating scan type:Extended；Confocality:Standard；Spectrum Range:Low 100.00；Centre Raman shit/cm‑1；High 2000.00；Configuration:Laser name 532nm edge；Grating name 1800l/mm；Exposure time/s:10.00；Accumulations:1；Objective:50；Laser power%:100；(2) detect；(3) Raman spectrogram is obtained；In the range of 500 2000 abscissas: be the peak of more than 4000 if there is more than 1 intensity, this Legionella to be measured is Legionella velogen strain；If being unsatisfactory for above-mentioned condition, this Legionella to be measured is Legionella low virulent strain.The method that the present invention provides is quickly, easy, testing cost is low.
Description
Technical field
The present invention relates to a kind of method for identifying Legionella virulence and dedicated kit thereof.
Background technology
Legionella, is aerobic gram-Negative bacillus, the most easily causes a disease with legionella pneumophilia.Existing 48 legions of Legionnella
Bacterial classification is found, and wherein 19 kinds are considered relevant with human airway infection.
Epidemiology survey confirm, Legionella by aerosol air-borne transmission to people, with cooling tower, hot water supply
System, steam inspissator, spa bathing pool, the clinical humidifier of breathing equipment, supermarket vegetables sprayer, fountain etc. have
Close.Indivedual reports, quote and also can be caused legionaires' disease by the water of legionella contaminated by the soil of legionella contaminated with contacting.It is at present
Only, interpersonal propagation has not proved out.The feature of legionaires' disease is that pneumonia accompanies general toxemia synptom, and severe patient goes out
Now loop exhaustion, respiratory failure around.X-ray findings of chest is single tikka sheet shade in early days, and pulmonary consolidation then, pathology is rapid
It is developed to many lobes of the lung section, pulmonary abscess can be had to be formed or a small amount of pleural effusion is levied.Make a definite diagnosis depend on specific antibody inspection and
Phlegm, pleural effusion, Lung biopsy sample isolate bacterium.
Summary of the invention
It is an object of the invention to provide a kind of method for identifying Legionella virulence and dedicated kit thereof.
The invention provides the kit of a kind of virulence identifying Legionella to be measured, including Raman spectrometer and record just like
The carrier of lower operational procedure:
(1) the following parameter of Raman spectrometer is set:
Key parameter is: Spectral acquisition setup；
Following parameter is set at Range interface:
Grating scan type:Extended；
Confocality:Standard；
Spectrum Range:Low 100.00；
Centre Raman shit/cm-1；
High 2000.00；
Configuration:Laser name 532nm edge；
Grating name 1800 l/mm；
Following parameter is set at Acquisition interface:
Exposure time/s:10.00；
Accumulations:1；
Objective:50；
Laser power%:100；
(2) take Legionella to be measured to be placed on slide, be placed in the Raman spectrometer of parameter setting of step (1)
Detect；
(3), after completing step (2), it is WiRE3.0 by the software design patterns that carries of Raman spectrometer, carries out data process,
To Raman spectrogram；In Raman spectrogram, in the range of 500-2000 abscissa: if there is more than 1 (containing 1) ordinate
(counts) intensity is the peak of more than 4000, and this Legionella to be measured is Legionella velogen strain；If being unsatisfactory for above-mentioned condition, this is treated
Surveying Legionella is Legionella low virulent strain.
The present invention also protects a kind of method of virulence identifying Legionella to be measured, comprises the steps:
(1) the following parameter of Raman spectrometer is set:
Key parameter is: Spectral acquisition setup；
Following parameter is set at Range interface:
Grating scan type:Extended；
Confocality:Standard；
Spectrum Range:Low 100.00；
Centre Raman shit/cm-1；
High 2000.00；
Configuration:Laser name 532nm edge；
Grating name 1800 l/mm；
Following parameter is set at Acquisition interface:
Exposure time/s:10.00；
Accumulations:1；
Objective:50；
Laser power%:100；
(2) take Legionella to be measured to be placed on slide, be placed in the Raman spectrometer of parameter setting of step (1)
Detect；
(3), after completing step (2), it is WiRE3.0 by the software design patterns that carries of Raman spectrometer, carries out data process,
To Raman spectrogram；In Raman spectrogram, in the range of 500-2000 abscissa: if there is more than 1 (containing 1) ordinate
(counts) intensity is the peak of more than 4000, and this Legionella to be measured is Legionella velogen strain；If being unsatisfactory for above-mentioned condition, this is treated
Surveying Legionella is Legionella low virulent strain.
Described Raman spectrometer concretely manufacturer is Reinshaw company of Britain (RENISHAW), and model is inVia
The Raman spectrometer of Raman Microscope.
Legionella velogen strain and Legionella low virulent strain are defined as follows: Legionella to be measured co-cultured with host cell, as
Fruit is compared with 0 moment co-cultured, and after co-culturing 24 hours, the Legionella quantity described to be measured in host cell increases, army to be measured
Group bacterium is Legionella intensity strain；If being unsatisfactory for above-mentioned condition, Legionella to be measured is Legionella low virulent strain.Described host cell has
Body can be mouse alveolar macrophages system J774.
The definition of Legionella velogen strain and Legionella low virulent strain is specific as follows: (1) takes Legionella to be measured, uses PBS
(pH7.2-7.4,0.01M) suspends, and obtains 108The bacteria suspension of cfu/ml, then dilutes bacteria suspension with RPMI1640 nutrient solution
To 10 times of volumes, obtaining concentration is 107The bacterium solution of cfu/ml；(2) RPMI1640 containing 10% (volume ratio) hyclone is used
Nutrient solution suspension mouse alveolar macrophages system J774, obtaining cell concentration is 2 × 105The cell suspension of individual cell/mL；(3)
Taking 24 orifice plates, every hole adds the cell suspension that 500 μ L step (2) obtain, and is placed in 37 DEG C, 5%CO2Environment is cultivated 4 hours；So
The bacterium solution that 1ml step (1) obtains is put in rear addition, is placed in 37 DEG C, 5%CO2Environment is cultivated 1.5 hours；Then take out 24 holes
Plate, cleans three times to remove non-adhering bacterium with PBS；Then every hole adds 1 milliliter of RPMI1640 nutrient solution, is placed in 37
DEG C, 5%CO2Co-culturing in environment, reject culture supernatant after co-culturing 0 hour or 24 hours, every hole adds 1mL sterilizing
Water, scrapes the cell bottom lower opening, and this cell suspension is transferred to the centrifuge tube of 1.5mL, is inoculated in after gradient dilution
BCYE flat board, carries out the colony counting of Legionella；If compared with 0 moment co-cultured, after co-culturing 24 hours every milliliter thin
In born of the same parents' suspension, the quantity of Legionella increases, and Legionella to be measured is Legionella intensity strain；If being unsatisfactory for above-mentioned condition, army to be measured
Group bacterium is Legionella low virulent strain.
The definition of Legionella velogen strain and Legionella low virulent strain is specific as follows: takes more than 5 BALB/c mouses, passes through tracheae
The mode sucked, it is 10 that every BALB/c mouse sucks 40 μ l bacterial contents8The bacteria suspension of the Legionella to be measured of cfu is to attack
Poison, attacks the 3rd day after poison, if the death rate of mouse be 100%, Legionella to be measured be Legionella velogen strain, if 60% with
Upper mouse has the morbidity of infection with legionella and characterizes and less than 60% dead mouse, Legionella to be measured are Legionella low virulent strain.
The method that the present invention provides, quickly, easy, need that sample size is few, testing cost is low, it is possible to identify that Legionella causes
Sick power is strong and weak, reduces the noise impact on measuring produced by veiling glare and background radiation, improves measurement accuracy.
Accompanying drawing explanation
Fig. 1 is the result figure of the step one of embodiment 1.
Fig. 2 is the result figure of the step 2 of embodiment 1.
Fig. 3 is the result figure of comparative example 1.
Fig. 4 is the result figure of comparative example 2.
Fig. 5 is the result figure of the step 2 of embodiment 2.
Fig. 6 is the result figure of the step 3 of embodiment 2.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is certainly
Routine biochemistry reagent shop is commercially available.Quantitative test in following example, is respectively provided with three times and repeats experiment, and result is made even
Average.The full name of ACES is 2-acetyl group-2-aminoethane sulphonic acid.
GVPC culture medium: glycine 3g/L, Polymyxin B sulfate 80000IU/L, vancomycin hydrochloride 0.001g/
L, cycloheximide 0.08g/L, YE (bacterium level) 10.0g, agar 12.0g, activated carbon 2.0g, KG
1.0g, ACES 10.0g, KOH 2.8g, L-cysteine hydrochloride 0.4g, ferric pyrophosphate 0.6g, distilled water is supplemented to
1000mL, adjusts pH value to 7.0 ± 0.2.
BCYE culture medium: YE (bacterium level) 10.0g, agar 12.0g, activated carbon 2.0g, KG
1.0g, ACES 10.0g, KOH 2.8g, L-cysteine hydrochloride 0.4g, ferric pyrophosphate 0.6g, distilled water is supplemented to
1000mL, adjusts pH value to 6.9.
BCYE-cys culture medium: YE (bacterium level) 10.0g, agar 12.0g, activated carbon 2.0g, α-one penta 2
Acid 1.0g, ACES10.0g, KOH 2.8g, ferric pyrophosphate 0.6g, distilled water is supplemented to 1000mL, adjusts pH value to 6.9.
Mouse alveolar macrophages system J774: exempt from Industrial Co., Ltd. purchased from upper sea base, article No. is clone J774.
BALB/c mouse: Beijing Vital River Experimental Animals Technology Co., Ltd., product article No. BALB/c, 7 week old.
The manufacturer of the Raman spectrometer used in embodiment is Reinshaw company of Britain (RENISHAW), and model is
inVia Raman Microscope。
Legionella is intracellular bacterial parasite.Legionella to be measured is co-cultured with host cell, if with the 0 moment phase co-cultured
Ratio, after co-culturing 24 hours, the Legionella quantity described to be measured in host cell increases, and Legionella to be measured is Legionella intensity strain；
If being unsatisfactory for above-mentioned condition, Legionella to be measured is Legionella low virulent strain.
The virulence of existing bacterial strain is compared by embodiment 1, the method for the employing present invention
Legionella (the Legionella pneumophila of numbered 33152 in Legionella ATCC33152, i.e. ATCC
Subsp.pneumophila Brenner et al.), velogen strain.
Legionella (the Legionella pneumophila of numbered 33156 in Legionella ATCC33156, i.e. ATCC
Subsp.fraseri Brenner et al.), velogen strain.
Legionella (the Legionella brunensis of numbered 43878 in Legionella ATCC43878 i.e. ATCC
Wilkinson et al.), low virulent strain.
Legionella (the Legionella spiritensis of numbered 35249 in Legionella ATCC35249 i.e. ATCC
Brenner et al.), low virulent strain.
Legionella (the Legionella cherrii Brenner of numbered 35252 in Legionella ATCC35252 i.e. ATCC
Et al), low virulent strain.
Legionella (the Legionella rubrilucens of numbered 35304 in Legionella ATCC35304 i.e. ATCC
Brenner et al.), low virulent strain.
Above 6 strain bacterium are proceeded as follows as test strains:
One, Raman spectrogram is obtained
1, test strains is seeded to BCYE culture medium, 37 DEG C, 110rpm shaken cultivation 18 hours, then 4 DEG C,
8000rpm is centrifuged 10 minutes, collects bacterial sediment.
2, the following parameter of Raman spectrometer be set:
Key parameter is: Spectral acquisition setup；
Following parameter is set at Range interface:
Grating scan type:Extended；
Confocality:Standard；
Spectrum Range:Low 100.00；
Centre Raman shit/cm-1；
High 2000.00；
Configuration:Laser name 532nm edge；
Grating name 1800 l/mm；
Following parameter is set at Acquisition interface:
Exposure time/s:10.00；
Accumulations:1；
Objective:50；
Laser power%:100.
3, it is placed in taking the bacterial sediment that a small amount of step 1 obtains on slide, has been placed into what the parameter of step 2 was arranged
Raman spectrometer detects.
4, after completing step 3, it is WiRE3.0 by the software design patterns that carries of Raman spectrometer, carries out data process, drawn
Graceful spectrogram.
The Raman spectrum of 6 test strains is shown in Fig. 1.Raman spectrogram according to 6 test strains is defined below judging mark
Accurate: at 500-2000 abscissa (Raman shift/cm-1In the range of): if there is more than 1 (containing 1) ordinate
(counts) intensity is the peak of more than 4000, and this Legionella to be measured is Legionella velogen strain；If being unsatisfactory for above-mentioned condition, this is treated
Surveying Legionella is Legionella low virulent strain.
Two, the virulence of each bacterial strain is verified by cell experiment
1, by test strains streak inoculation to BCYE culture medium flat plate, it is placed in 37 DEG C, 5%CO2Environment is cultivated 48 hours.
2, after completing step 1, the bacterium on scraping flat board, suspend with PBS (pH7.2-7.4,0.01M), obtain
108The bacteria suspension of cfu/ml, is then diluted to 10 times of volumes with RPMI1640 nutrient solution by bacteria suspension, and obtaining concentration is 107cfu/
The bacterium solution of ml.
3, the RPMI1640 nutrient solution suspension mouse alveolar macrophages system containing 10% (volume ratio) hyclone is used
J774, obtaining cell concentration is 2 × 105The cell suspension of individual cell/mL.
4, taking 24 orifice plates, every hole adds the cell suspension that 500 μ L steps 3 obtain, and is placed in 37 DEG C, cultivates in 5%CO2 environment
4 hours；It is subsequently adding and puts into the bacterium solution that 1ml step 2 obtains, be placed in 37 DEG C, 5%CO2Environment is cultivated 1.5 hours；Then take
Go out 24 orifice plates, clean three times to remove non-adhering bacterium with PBS；Then every hole adds 1 milliliter of RPMI1640 nutrient solution,
It is placed in 37 DEG C, 5%CO2Environment is cultivated, respectively at cultivating 0 hour, 24 hours, after 48 hours or 72 hours in reject cultivation
Clearly, every hole adds 1mL aqua sterilisa, scrapes with rubber cell, scrapes the cell bottom lower opening, and be transferred to by this cell suspension
The centrifuge tube of 1.5mL, is inoculated in BCYE flat board after gradient dilution, carry out the colony counting of Legionella.
Result is shown in Fig. 2 (repeating the mean value of experiment for three times), and ordinate is the number of Legionella in every milliliter of cell suspension
Amount.
Three, the virulence of each existing bacterial strain is verified by zoopery
BALB/c mouse is divided into 7 groups, often group 10, attacks poison the most as follows and process:
First group: by the way of tracheae sucks, every mouse sucks bacteria suspension (its of 40 μ l Legionella ATCC33152
Middle Legionella bacterial content is 108Cfu) to attack poison；
Second group: by the way of tracheae sucks, every mouse sucks bacteria suspension (its of 40 μ l Legionella ATCC33156
Middle Legionella bacterial content is 108cfu)；
3rd group: by the way of tracheae sucks, every mouse sucks bacteria suspension (its of 40 μ l Legionella ATCC43878
Middle Legionella bacterial content is 108Cfu) to attack poison；
4th group: by the way of tracheae sucks, every mouse sucks bacteria suspension (its of 40 μ l Legionella ATCC35249
Middle Legionella bacterial content is 108Cfu) to attack poison；
5th group: by the way of tracheae sucks, every mouse sucks bacteria suspension (its of 40 μ l Legionella ATCC35252
Middle Legionella bacterial content is 108Cfu) to attack poison；
6th group: by the way of tracheae sucks, every mouse sucks bacteria suspension (its of 40 μ l Legionella ATCC35304
Middle Legionella bacterial content is 108Cfu) to attack poison；
7th group (blank group): by the way of tracheae sucks, every mouse sucks 40 μ l physiological saline spray liquids.
Attack the 3rd day after poison, the number of elements of statistics often group morbidity mouse and death condition.Morbidity is characterized as below: reaction is late
Slow and shake all over and One's spirits are drooping and motion is smooth and with fervescence.
First group of mouse with morbidity sign is 10 (10 equal death), and second group of mouse with morbidity sign is
10 (10 equal death), the 3rd group of mouse with morbidity sign is 8 (not having dead mouse), and the 4th group has morbidity table
The mouse levied is 8 (not having dead mouse), and the 5th group of mouse with morbidity sign is 7 (wherein 1 death), the 6th group
The mouse with morbidity sign is 9 (not having dead mouse), the 7th group of sign of the most not falling ill.
Comparative example 1,
Legionella (the Legionella pneumophila of numbered 33152 in Legionella ATCC33152, i.e. ATCC
Subsp.pneumophila Brenner et al.), velogen strain.
Legionella (the Legionella pneumophila of numbered 33156 in Legionella ATCC33156, i.e. ATCC
Subsp.fraseri Brenner et al.), velogen strain.
Legionella (the Legionella brunensis of numbered 43878 in Legionella ATCC43878 i.e. ATCC
Wilkinson et al.), low virulent strain.
Legionella (the Legionella spiritensis of numbered 35249 in Legionella ATCC35249 i.e. ATCC
Brenner et al.), low virulent strain.
Above 4 strain bacterium are proceeded as follows as test strains:
1, test strains is seeded to BCYE culture medium, 37 DEG C, 110rpm shaken cultivation 18 hours, then 4 DEG C,
8000rpm is centrifuged 10 minutes, collects bacterial sediment.
2, the following parameter of Raman spectrometer be set:
Configuration:Laser name 1320nm edge；
Other parameters all with embodiment 1 step one 2 in parameter.
The Raman spectrum of 4 test strains is shown in Fig. 3.
Comparative example 2,
Legionella (the Legionella pneumophila of numbered 33152 in Legionella ATCC33152, i.e. ATCC
Subsp.pneumophila Brenner et al.), velogen strain.
Legionella (the Legionella pneumophila of numbered 33156 in Legionella ATCC33156, i.e. ATCC
Subsp.fraseri Brenner et al.), velogen strain.
Legionella (the Legionella brunensis of numbered 43878 in Legionella ATCC43878 i.e. ATCC
Wilkinson et al.), low virulent strain.
Legionella (the Legionella spiritensis of numbered 35249 in Legionella ATCC35249 i.e. ATCC
Brenner et al.), low virulent strain.
Above 4 strain bacterium are proceeded as follows as test strains:
1, test strains is seeded to BCYE culture medium, 37 DEG C, 110rpm shaken cultivation 18 hours, then 4 DEG C,
8000rpm is centrifuged 10 minutes, collects bacterial sediment.
2, the following parameter of Raman spectrometer be set:
Exposure time/s:100.00；
Other parameters all with embodiment 1 step one 2 in parameter.
The Raman spectrum of 4 test strains is shown in Fig. 4.
The virulence of the bacterial strain of collection is compared by embodiment 2, the method for the employing present invention
One, bacterial strain is gathered
In March, 2014, the sewer from Beijing gathers water sample.
The method separating Legionella from water sample is as follows:
1, water sample is coated GVPC culture medium flat plate, be placed in 35 DEG C, 2.5%CO2Incubator is cultivated 48 hours.
2, after completing step 1, the bacterium colony on picking flat board, use BCYE culture medium flat plate, be placed in 35 DEG C, 2.5%CO2Training
Support in case and cultivate, use dilution plate method, obtain pure bacterial strain.
3, each pure bacterial strain step 2 obtained carries out Morphological Identification respectively, meet following morphological feature for candidate's
Legionella (Legionella growth is slow, is easily covered by other bacterium, needs observe on body formula mirror every day): colony colour is various, generally
White, grey, blueness or purple, also can show dark brown, celadon, peony；Bacterium colony is neat, smooth surface, in typical case's hair
Glassy；Fluorescence is had under uviol lamp.
4, the Legionella of each candidate step 3 obtained is verified the most as follows: inoculation test strains is inoculated respectively
To BCYE culture medium flat plate and BCYE-cys culture medium, it is placed in 35 DEG C, 2.5%CO2Incubator is cultivated 2 days, if bacterium to be measured
Strain grows on BCYE culture medium flat plate and does not grows on BCYE-cys culture medium flat plate, and this bacterial strain is the Legionella of candidate.
5, the Legionella of each candidate obtained of step 4 is verified the most further, if meeting following bar
Part, this bacterial strain is Legionella: oxidizing ferment (-/weak+), nitrate reduction-, urease-, gelatin liquefaction+, hydrolyze hippuric acid.
Obtain 3 strain Legionella altogether.
Two, the Raman spectrogram of bacterial strain is obtained
Step one with embodiment 1.
The Raman spectrogram of the 3 strain Legionella that step one obtains is shown in Fig. 5.Strain 1 and strain 2 are Legionella velogen strain, poison
Strain 3 is Legionella low virulent strain.
Three, the virulence of each existing bacterial strain is verified by cell experiment
Method is with the step 2 of embodiment 1.
The virulence of the 3 strain Legionella that step one obtains is shown in Fig. 6.Strain 1 and strain 2 are Legionella velogen strain, and strain 3 is army
Group's bacterium low virulent strain.
Four, the virulence of each existing bacterial strain is verified by zoopery
BALB/c mouse is divided into 7 groups, often group 10, attacks poison the most as follows and process:
First group: by the way of tracheae sucks, every mouse sucks bacteria suspension (the wherein Legionella bacterium of 40 μ l strains 1
Content is 108Cfu) to attack poison；
Second group: by the way of tracheae sucks, every mouse sucks bacteria suspension (the wherein Legionella bacterium of 40 μ l strains 2
Content is 108Cfu) to attack poison；
3rd group: by the way of tracheae sucks, every mouse sucks bacteria suspension (the wherein Legionella bacterium of 40 μ l strains 3
Content is 108Cfu) to attack poison；
4th group (blank group): by the way of tracheae sucks, every mouse sucks 40 μ l physiological saline spray liquids.
Attack the 3rd day after poison, the number of elements of statistics often group morbidity mouse and death condition.Morbidity is characterized as below: reaction is late
Slow and shake all over and One's spirits are drooping and motion is smooth and with fervescence.
First group of mouse with morbidity sign is 10 (10 equal death), and second group of mouse with morbidity sign is
10 (10 equal death), the 3rd group of mouse with morbidity sign is 7 (not having dead mouse), does not the most fall ill for the 4th group
Characterize.
Claims (1)
1. the method identifying the virulence of Legionella to be measured, comprises the steps:
(1) the following parameter of Raman spectrometer is set:
Key parameter is: Spectral acquisition setup；
Following parameter is set at Range interface:
Grating scan type:Extended；
Confocality:Standard；
Spectrum Range:Low 100.00；
Centre Raman shit/cm-1；
High 2000.00；
Configuration:Laser name 532nm edge；
Grating name 1800l/mm；
Following parameter is set at Acquisition interface:
Exposure time/s:10.00；
Accumulations:1；
Objective:50；
Laser power%:100；
(2) take Legionella to be measured to carry out pretreatment and be placed on slide, be placed into the Raman that the parameter of step (1) is arranged
Spectrometer detects；The method of described pretreatment is: Legionella to be measured is seeded to BCYE culture medium, 37 DEG C, 110rpm
Shaken cultivation 18 hours, then 4 DEG C, 8000rpm be centrifuged 10 minutes, collect bacterial sediment；
(3), after completing step (2), it is WiRE3.0 by the software design patterns that carries of Raman spectrometer, carries out data process, drawn
Graceful spectrogram；In Raman spectrogram, in the range of 500-2000 abscissa: be 4000 if there is more than 1 ordinate intensity
Above peak, this Legionella to be measured is Legionella velogen strain；If being unsatisfactory for above-mentioned condition, this Legionella to be measured is that Legionella is weak
Strain；
Legionella velogen strain and Legionella low virulent strain are defined as follows: Legionella to be measured co-cultured with host cell, if with
0 moment co-cultured is compared, and after co-culturing 24 hours, the Legionella quantity described to be measured in host cell increases, Legionella to be measured
For Legionella intensity strain；If being unsatisfactory for above-mentioned condition, Legionella to be measured is Legionella low virulent strain.
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