CN107505304A - A kind of quick method for judging drug-resistant type Candida albicans - Google Patents
A kind of quick method for judging drug-resistant type Candida albicans Download PDFInfo
- Publication number
- CN107505304A CN107505304A CN201710618991.3A CN201710618991A CN107505304A CN 107505304 A CN107505304 A CN 107505304A CN 201710618991 A CN201710618991 A CN 201710618991A CN 107505304 A CN107505304 A CN 107505304A
- Authority
- CN
- China
- Prior art keywords
- candida albicans
- drug
- resistant type
- spectrum
- raman
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
Landscapes
- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to medicine analysis field, specifically a kind of quick method for judging drug-resistant type Candida albicans, comprise the following steps：Step 1：Dynamic surface strengthens the collection of Raman spectrum；Step 2：The pretreatment of spectrum；Step 3：The Candida albicans of unknown susceptibility is judged than scope according to the peak intensity at characteristic peak and top.The invention has the advantages that：Step is simple, and technical solution of the present invention to sample without being destroyed and being taken shorter；High sensitivity, compared with normal Raman spectroscopic methodology even conventional surface enhancing Raman spectroscopy, it is capable of detecting when the determinand of lower loading；Differentiate simply and be more applicable for a clinical line.
Description
Technical field
The present invention relates to medicine analysis technical field, is a kind of quick judgement drug-resistant type Candida albicans specifically
Method.
Background technology
In recent years, a large amount of abuses of antifungal drug result in increasingly severe Antifungal resistance.Candida albicans, make
For most common fungi, infection caused by its caused drug-resistant type bacterial strain directly threatens the life of patient.Therefore, one kind is needed badly
The quick method for judging drug-resistant type Candida albicans.
Traditional cell biology method includes enzyme linked immunological group, PCR etc., and these methods need multiple
Miscellaneous sample pre-treatments, cytoclasis need to such as be extracted albumen or nucleotide sequence therein and be analyzed.And relatively simple medicine
Although quick test method(s) need not destroy cell, continue to seem time-consuming longer with medicine culture 24h even 48h after 16h is passed on.It is comprehensive
On, these methods quickly and simply can not be judged drug-resistant type Candida albicans.
Raman spectroscopy is due to possessing the spy almost without sample treatment (without destroying cell), quick detection (a few minutes)
Point, it is typically used to the discriminating and differentiation of microorganism recent years.But it is faint due to Raman signal, cause the differentiation of this method
The degree of accuracy is inadequate.Need to expend the sample of larger dosage to enhancing signal.And SERS (SERS) technology because
(relative to the Raman spectrum of routine, SERS signal can increase by several orders of magnitude to its higher detection sensitivity, therefore only need
Want less cell dosage), this defect can be overcome.Characteristic peak more abundant will after spectral signal is strengthened simultaneously
It is more beneficial for distinguishing the raising of the degree of accuracy.Generally we are judging that drug-resistant type reads a plant bacterium in vain using SERS method
When, reference is used as using responsive type Candida albicans.And responsive type and drug-resistant type Candida albicans produce during subculture
Raw material composition variation is extremely small, and the mostly just peak position identification with some characteristic peak is difficult to responsive type and resistance
Type Candida albicans is distinguished.
The deficiency shown for the above method in differentiation responsive type and drug-resistant type, Chemical Measurement method such as clustering methodology,
PCA, partial least squares discriminant analysis method etc. be used to amplify the further processing of spectral signal trickle difference
It is different.But these methods need substantial amounts of data, and handle these data and need to write program code.These steps are for clinic
Seem excessively complicated for testing staff, be not suitable for actual utilization.And if the method for passing through these Chemical Measurements
Searching out the peak intensity at several feature SERS peaks, when corresponding discrimination standard can then seem and very simple and be more applicable for facing
One line of bed.
The content of the invention
It is an object of the invention to by finding out above-mentioned said several feature peak intensity ratios come white to responsive type and drug-resistant type
Color candida albicans carries out quick, simple, accurately differentiation, so as to realize the Early judgement to drug-resistant type Candida albicans.
To achieve these goals, a kind of quick method for judging drug-resistant type Candida albicans of present invention offer, including with
Lower step：
Step 1：Dynamic surface strengthens the collection of Raman spectrum
Candida albicans to be measured is mixed with isometric surface enhanced reagent, takes mixing drop on silicon chip；Using drawing
Graceful spectrometer, continuous Raman spectrum collection is carried out to the drop on silicon chip, until drop evaporates into dry state by hygrometric state, obtain one
The one-dimensional dynamic SERS figure of series；
Step 2：The pretreatment of spectrum
The spectrogram obtained to step 1 pre-processes, and pretreatment includes choosing 600~1800cm-1Spectral region,
Selection range interior focusing spectrum carries out airPLS method baseline corrections, and Whittaker-Smooth methods are smooth；Every plant is chosen after pre-processing
The maximum intensity that bacterium continuous acquisition obtains is (with top 656cm-1For standard) spectrum carry out follow-up differentiation；
Step 3：The Candida albicans of unknown susceptibility is judged than scope according to the peak intensity at characteristic peak and top
Pass through following 3 characteristic peaks, respectively 724cm-1、1094cm-1、1465cm-1With top 656cm-1Ratio
And corresponding discrimination standard, it is responsive type or drug-resistant type to identify Candida albicans to be measured；Its criterion is：I724/I656>
0.6 or I1094/I656>0.13 or I1465/I656>0.24, then it is determined as drug-resistant type Candida albicans；I724/I656<0.4 or I1094/
I656<0.09 or I1465/I656<0.22, then it is determined as responsive type Candida albicans.
Preferably, the Candida albicans to be measured in described step one is passage 16-20h Candida albicans.
Preferably, 1-3 μ L mixing drops are taken in described step one on silicon chip.
Preferably, laser intensity is 100-300mW, acquisition time 1-10s in described step one.
Preferably, every plant of bacterium gathers 5-10 times in described step one.
Compared with prior art, the advantages of technical scheme and good effect is as follows：
1st, step is simple, takes shorter
Conventional molecular biology method need to carry out complicated pre-treatment to sample, and such as cell is destroyed, rear separation
Extraction related substances is analyzed again.Though drug sensitive test method need not destroy cell, still needing to time-consuming 24-48h can just enter to result
Row judges.And technical solution of the present invention need not be destroyed to sample and take shorter (only a few minutes), this obviously can expire
The quick diagnostic requirements for judging analysis of clinic pathogenic microorganism of foot.
2nd, high sensitivity
Technical solution of the present invention is using dynamic surface enhancing Raman spectroscopy.With the volatilization of liquid, focus largely produces
Raw, the spectral signal for the cell to be measured being placed in focus has obtained great enhancing (improving several orders of magnitude).With conventional drawing
Graceful spectroscopic methodology even conventional surface enhancing Raman spectroscopy is compared, and is capable of detecting when the determinand of lower loading.
3rd, differentiate simply and be more applicable for a clinical line
The conventional method of discrimination based on Raman spectrum overall signal, typically using PCA, offset minimum binary
The stoechiometric process such as techniques of discriminant analysis, further to handle or amplify trickle SPECTRAL DIVERSITY.But these methods depend on
Substantial amounts of sample and spectroscopic data, and software program.Characteristic peak proposed by the present invention than method, independent of substantial amounts of sample
Sheet and spectroscopic data, or software program, differentiation is simpler, is more suitable for a clinical line and uses.
Brief description of the drawings
Fig. 1 is that differentiation responsive type and drug-resistant type read a plant flow chart for bacterium decision method in vain in embodiment 1；
Fig. 2 is that the dynamic surface of one plant of Candida albicans in embodiment 1 strengthens Raman spectrogram；
2A is the SERS variation diagram of a series of continuous acquisition；2B is top 656cm-1Peak intensity become
Change figure.
Fig. 3 is that the white continuous dynamic surface of strain bacterium of reading of 20 plants in embodiment 1 strengthens the spectrum of maximum intensity in Raman spectrum
Figure；
Fig. 4 is the full spectral limit (600-1800cm in embodiment 1-1) principal component load diagram；
Fig. 5 is the principal component scores figure in the characteristic spectrum section in embodiment 1；
5A is spectral region 690-740cm-1；5B is spectral region 950-1000cm-1；5C is spectral region 1070-
1120cm-1；5D is spectral region 1300-1350cm-1；5E is spectral region 1450-1500 cm-1；5F is spectral region 1670-
1720cm-1；
Fig. 6 is 3 characteristic peaks of 20 plants of Candida albicans and the peak intensity ratio at top in embodiment 1.
Embodiment
Embodiment provided by the invention is elaborated with reference to embodiment.
Embodiment 1
The Raman spectrometer that embodiment 1 uses is K-sens Raman spectrum systems (Shanghai Fu Xiang Science and Technology Ltd.), is swashed
Send out wavelength 785nm.For nano silver colloidal sol, preparation method is the surface enhanced reagent used：Take AgNO336mg add it is secondary go from
Sub- water 200mL dissolvings, are heated to micro-boiling.1% trisodium citrate aqueous solution 4mL is added dropwise, and is stirred vigorously, is persistently added
Hot 30min.Celadon colloid is obtained, is cooled to room temperature, it is standby.
Fig. 1 is to read a plant flow chart for bacterium decision method in vain to distinguishing responsive type and drug-resistant type in embodiment 1, specifically include with
Lower three steps：
S1：Dynamic surface strengthens the collection of Raman spectrogram
By pass on 16h 10 plants of responsive types and 10 plants of drug-resistant type Candida albicans respectively with isometric surface enhanced reagent
Mixing, takes 3 μ L mixing drops on silicon chip.Using Raman spectrometer, 300mW laser intensity, time of integration 5s, spectrum are chosen
Acquisition time interval 0s, continuous Raman spectrum collection is carried out to the drop on silicon chip, until drop evaporates into dry state by hygrometric state,
Obtain a series of one-dimensional dynamic SERS figures (Fig. 2), respectively collection 6 times of every plant of bacterium.
S2：Analysis of the PCA to spectrum
Using the preprocessor of Matlab software programmings, collection of illustrative plates obtained by step 1 is pre-processed, including chosen
600-1800cm-1Spectral region, composed in selection range interior focusing and carry out airPLS method baseline corrections, Whittaker-Smooth
Method is smooth.Choose spectrum (every plant of bacterium chooses 6 spectrum) (figure of maximum peak intensity in a series of spectrum of every plant of bacterium continuous acquisition
3).120 spectrum of selection are standardized using the normalization program of Matlab software programmings.Recycle Matlab soft afterwards
The principal component analysis program that part is write carries out that corresponding principal component load diagram (Fig. 4) is calculated to these spectrum.Select load
VIP values in figure>0.05 characteristic peak, with the characteristic spectrum section (50cm where 6 characteristic peaks of selection-1) led again
Constituent analysis calculates, and obtains the principal component scores figure (Fig. 5) in each characteristic spectrum section.As a result wherein three characteristic peaks are shown
724cm-1、1094cm-1、1465cm-1Representative characteristic spectrum section shot chart PC1+PC2 is all higher than 90% and by a certain
Boundary can distinguish responsive type and drug-resistant type Candida albicans.
S3：Read according to the peak intensity at the above-mentioned characteristic peak found out and top than scope to distinguish responsive type and drug-resistant type white
Pearl bacterium.
By S2, we have found 3 characteristic peaks for meeting above-mentioned condition, respectively 724cm-1、1094 cm-1、
1465cm-1.This 3 characteristic peaks found out by above-mentioned principal component analysis are to distinguish responsive type and drug-resistant type Candida albicans
Important symbol.In order to eliminate the influence of peak intensity, we calculate above three characteristic peak and highest peak 656cm respectively-1Peak intensity ratio,
Its result is as shown in Figure 6:I724/I656>0.6 or I1094/I656>0.13 or I1465/I656>0.24, then it is determined as drug-resistant type white
Candida albicans；I724/I656<0.4 or I1094/I656<0.09 or I1465/I656<0.22, then it is determined as responsive type Candida albicans.
Embodiment 2
A kind of quick method for judging drug-resistant type Candida albicans of the present invention, specifically includes following steps：
Step 1：Dynamic surface strengthens the collection of Raman spectrum
The Candida albicans of the more plants of unknown susceptibilitys of certain time (16-20h) and isometric surface enhanced will be passed on
Reagent mixes, and takes certain volume mixing drop on silicon chip.Using Raman spectrometer, certain laser intensity, the time of integration are chosen,
Continuous Raman spectrum collection is carried out to the drop on silicon chip, until drop evaporates into dry state by hygrometric state, obtained a series of one-dimensional
Dynamic surface strengthens Raman spectrogram.Certain volume is 1-3 μ L, laser intensity 100-300mW, acquisition time 1-10s.Often
Strain bacterium respectively gathers 5-10 times.
Step 2：The pretreatment of spectrum
All collection of illustrative plates obtained to step 1 pre-process, and pretreatment includes choosing 600~1800cm-1Spectral region,
Composed in selection range interior focusing and carry out airPLS method baseline corrections, Whittaker-Smooth methods are smooth.Choose every after pre-processing
The maximum intensity that strain bacterium continuous acquisition obtains is (with top 656cm-1For standard) spectrum carry out follow-up differentiation.
Step 3：The Candida albicans of unknown susceptibility is judged than scope according to the peak intensity at characteristic peak and top
By following 3 characteristic peaks, respectively 724,1094,1465cm-1With top 656cm-1Ratio and corresponding
Discrimination standard, the Candida albicans for identifying unknown susceptibility is responsive type or drug-resistant type by we.The following institute of its criterion
Show:I724/I656>0.6 or I1094/I656>0.13 or I1465/I656>0.24, then it is determined as drug-resistant type Candida albicans；I724/I656<
0.4 or I1094/I656<0.09 or I1465/I656<0.22, then it is determined as responsive type Candida albicans.
The inventive method detects accuracy rate during unknown strains>80%.
The preferred embodiment to the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (5)
- A kind of 1. quick method for judging drug-resistant type Candida albicans, it is characterised in that comprise the following steps：Step 1：Dynamic surface strengthens the collection of Raman spectrumCandida albicans to be measured is mixed with isometric surface enhanced reagent, takes mixing drop on silicon chip；Using Raman light Spectrometer, continuous Raman spectrum collection is carried out to the drop on silicon chip, until drop evaporates into dry state by hygrometric state, obtained a series of One-dimensional dynamic SERS figure；Step 2：The pretreatment of spectrumThe spectrogram obtained to step 1 pre-processes, and pretreatment includes choosing 600~1800cm-1Spectral region, choosing In the range of airPLS method baseline corrections are carried out to spectrum, Whittaker-Smooth methods are smooth；Choose white to be measured after pre-processing The spectrum for the maximum intensity that candida albicans continuous acquisition obtains carries out follow-up differentiation, and maximum intensity is with top 656cm-1For mark It is accurate；Step 3：The Candida albicans of unknown susceptibility is judged than scope according to the peak intensity at characteristic peak and topPass through following 3 characteristic peaks, respectively 724cm-1、1094cm-1、1465cm-1With top 656cm-1Ratio and corresponding Discrimination standard, it is responsive type or drug-resistant type to identify Candida albicans to be measured；Its criterion is：I724/I656>0.6 or I1094/I656>0.13 or I1465/I656>0.24, then it is determined as drug-resistant type Candida albicans；I724/I656<0.4 or I1094/I656< 0.09 or I1465/I656<0.22, then it is determined as responsive type Candida albicans.
- 2. the method for drug-resistant type Candida albicans is quickly judged according to claim 1, it is characterised in that in described step one Candida albicans to be measured is passage 16-20h Candida albicans.
- 3. the method for drug-resistant type Candida albicans is quickly judged according to claim 1, it is characterised in that taken in described step one 1-3 μ L mixing drops are on silicon chip.
- 4. the method for drug-resistant type Candida albicans is quickly judged according to claim 1, it is characterised in that swash in described step one Luminous intensity is 100-300mW, acquisition time 1-10s.
- 5. the method for drug-resistant type Candida albicans is quickly judged according to claim 1, it is characterised in that every in described step one Strain bacterium gathers 5-10 times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710618991.3A CN107505304A (en) | 2017-07-26 | 2017-07-26 | A kind of quick method for judging drug-resistant type Candida albicans |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710618991.3A CN107505304A (en) | 2017-07-26 | 2017-07-26 | A kind of quick method for judging drug-resistant type Candida albicans |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107505304A true CN107505304A (en) | 2017-12-22 |
Family
ID=60690016
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710618991.3A Pending CN107505304A (en) | 2017-07-26 | 2017-07-26 | A kind of quick method for judging drug-resistant type Candida albicans |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107505304A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872187A (en) * | 2018-03-30 | 2018-11-23 | 詹晓凯 | A kind of method and its application using Raman image technology detection drug resistance |
| CN109781702A (en) * | 2019-01-18 | 2019-05-21 | 中国人民解放军军事科学院军事医学研究院 | A kind of detection method of magnetic microsphere and preparation method thereof and microorganism |
| CN111624190A (en) * | 2020-06-11 | 2020-09-04 | 复旦大学附属华山医院 | Method for rapidly identifying bacteria and fungi by using Raman spectrum |
| CN113390853A (en) * | 2021-07-21 | 2021-09-14 | 中国科学院苏州生物医学工程技术研究所 | Method and system for identifying bacteria and fungi by using Raman spectrum |
| WO2024099394A1 (en) * | 2022-11-09 | 2024-05-16 | 清华大学 | Surface-enhanced raman scattering file card and manufacturing method therefor, and method for performing quantitative analysis by using file card |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106841168A (en) * | 2017-03-28 | 2017-06-13 | 北京工业大学 | A kind of SERS method for detecting Salmonella typhimurium drug-fast bacteria and non-drug-fast bacteria in identification sample |
-
2017
- 2017-07-26 CN CN201710618991.3A patent/CN107505304A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106841168A (en) * | 2017-03-28 | 2017-06-13 | 北京工业大学 | A kind of SERS method for detecting Salmonella typhimurium drug-fast bacteria and non-drug-fast bacteria in identification sample |
Non-Patent Citations (1)
| Title |
|---|
| 陆峰 等: ""耐药型白念珠菌的红外光谱判别方法的初步研究"", 《分析化学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872187A (en) * | 2018-03-30 | 2018-11-23 | 詹晓凯 | A kind of method and its application using Raman image technology detection drug resistance |
| CN109781702A (en) * | 2019-01-18 | 2019-05-21 | 中国人民解放军军事科学院军事医学研究院 | A kind of detection method of magnetic microsphere and preparation method thereof and microorganism |
| CN111624190A (en) * | 2020-06-11 | 2020-09-04 | 复旦大学附属华山医院 | Method for rapidly identifying bacteria and fungi by using Raman spectrum |
| CN113390853A (en) * | 2021-07-21 | 2021-09-14 | 中国科学院苏州生物医学工程技术研究所 | Method and system for identifying bacteria and fungi by using Raman spectrum |
| WO2024099394A1 (en) * | 2022-11-09 | 2024-05-16 | 清华大学 | Surface-enhanced raman scattering file card and manufacturing method therefor, and method for performing quantitative analysis by using file card |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107505304A (en) | A kind of quick method for judging drug-resistant type Candida albicans | |
| CN105181912B (en) | A kind of Noninvasive Measuring Method of Freshness in rice storage | |
| CN105219844B (en) | Gene marker combination, kit and the disease risks prediction model of a kind of a kind of disease of screening ten | |
| CN109001180A (en) | A kind of Raman spectrum combination artificial intelligence high throughput single cell analysis identification method | |
| CN110160968A (en) | Failure or penalty with fluorescent material detection fluorimeter | |
| CN111458314B (en) | Method for identifying production place of dried orange peel based on autofluorescence-quenching fluorescence splicing spectrum | |
| US20210389243A1 (en) | System and method for quantification of bacteria in water using fluorescence spectra measurements and machine-learning | |
| US20200326285A1 (en) | Detection and analysis method for urine-modified nucleoside based on surface-enhanced resonance raman spectroscopy | |
| CN106520977A (en) | Primers and method for detecting alfalfa root rot fungi by virtue of loop-mediated isothermal amplification | |
| CN105954412A (en) | Sensor array optimization method for Carya cathayensis freshness detection | |
| CN107741439A (en) | A kind of evaluation method of the mellow and full property of beer wine body | |
| CN112730399A (en) | Method for rapidly detecting white spirit based on nano material-organic dye colorimetric sensor array | |
| CN109913565A (en) | A kind of kit for detecting vibrio parahaemolytious, primer pair, probe and method | |
| Peijin et al. | Rapid identification of the variety and geographical origin of Wuyou No. 4 rice by fourier transform near-infrared spectroscopy coupled with chemometrics | |
| US9316591B1 (en) | Biosensor for detection of subclinical ketosis | |
| CN106338488A (en) | Method for fast undamaged determination of transgenic soybean milk powder | |
| CN107727711A (en) | A kind of evaluation method of beer wine body full weight | |
| US20060234319A1 (en) | Disease determination method, data generation method for disease determination and data generation system for disease determination | |
| Mizrach et al. | Yeast detection in apple juice using Raman spectroscopy and chemometric methods | |
| CN108802163A (en) | A method of different honey categories of quickly reflecting | |
| US20090053751A1 (en) | Chemiluminescent Method and Device for Evaluating the In Vivo Functional State of Phagocytes | |
| CN110257544A (en) | Ergot germ fluorescence quantitative PCR detection reagent and detection kit and application | |
| CN112143178B (en) | Electrostatic composite fluorescent array sensor and application | |
| CN102952851B (en) | Primer for detecting kind and/or purity of malting barley, and kit and method thereof | |
| CN108344709A (en) | A method of quickly differentiating potato sprouting characteristic based on near-infrared spectrum technique |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171222 |