|Title of Invention||
AN ASSAY STRIP FOR DETECTION OF SIDEROPHORE IN A SAMPLE
|Abstract||The present invention is directed toward a rapid and easy detection of microbial siderophores using dye reagent. To know the iron acquisition systems the detection and determination of siderophores are absolutely crucial and peremptory requirement of microbial system. Variety of methods were proposed and used, however, the ultimate use at field remains tricky and time consuming and hence, demand simple and handy toot. The invention described herein can facilitate the easy detection of siderohpore of possible biomedical and agricultural interest. The present process using strips shows maximum specificity of assay toward microbial siderophore under different parameters than any of the method available system.|
|Full Text||FORM 2
THE PATENT ACT 1970
(39 OF 1970)
THE PATENTS RULES, 2003
TITLE OF THE INVENTION
A METHOD FOR DETECTION OF SIDEROPHORE USING DYE PAPER STRIP
Mr. Kadam Meghraj Shaligram
Dr, Ambalal Babulal Chaudhari
Prof. Sudhir Bhaskarrao Chincholkar
A METHOD FOR DETECTION OF SIDEROPHORE USING DYE PAPER STRIP
The following specification particularly describes the invention and the manner in which it is to be performed
FIELD OF THE INVENTION
The invention relates to a method for development of dye impregnated paper strip for detection of microbial siderophore. More specifically, the invention relates to a process of dye impregnated strip production and analyzing effect of different parameters for specificity and sensitivity toward microbial siderophore.
BACKGROUND OF THE INVENTION
Under iron tress condition, various microorganisms produce and secrete a variety
of low molecular weight (500-1000 Da) and high affinity chelating agents termed
siderophore, which solubilize ferric iron in environment and transport it into the
cell (A. Machuca and A.M.F. Milagres, 2003). Siderophores are typically
produced by bacteria, fungi, and monocotyledonous plants. Siderophores exhibit
considerable structural variability and affinity for iron e.g., aerobactin. Microbial
siderophores are basically catecholates and hydroxamates type (Perez-Miranda,
2007). Besides microbial iron nutrition, many siderophores play important role in
(i) microbial infection, (ii) the antagonism of Plant Growth Promoting
Rhizobacteria (PGPR) against plant pathogens (Xiaojun Xie, 2006) and (iii) medical application (desferal- a siderophore produced by Streptomyces sp.). A wide variety of methods are available to detect siderophores viz. chemical methods, like Csaky test and colorimetric ferric perchlorate assay to detect hydroxamate-type of siderophores whereas, Arnow test to detect catechol type siderophores (Manninen and Mattila-Sandholm, 1994). Besides, electrophoresis, high-voltage paper chromatography and fluorescence under UV light have been
used to detect siderophores in spent media (Guillermo Carrillo-Castan'edaa, 2005).
Of these, universal chemical assay consisting of chrome azurol sulfonate (CAS) is most widely used for detection of siderophores due to its sensitivity, reproducibility and convenience. The ternary complex CAS/Fe (lll)/hexadecyl trimethyl ammonium bromide serves as an indicator. The color of the ferric complexes generally range from yellow-brown to red-brown depending on the nature of the ligands, apart from blue coloration of CAS indicator (Bernhard Schwyn and J B Nelands, 1987; Heiko Hayen and Dietrich A. Volmer, 2005). Siderophore detection with isoelectric focusing followed by subsequent fluorescence under UV illumination or iron chelation through CAS overlay (Nico Koedam, 1994) remains an alternative method.
In summary, CAS assay has been preferred as a solution test to measure siderophores produced by microbes in chemically defined media. This assay is partially affected by yellowish ingredients of complex culture media and supplemented biological fluids. Alternative CAS assay consist of CAS agar plate overlaid with CAS reagent and CAS agar diffusion method. Major drawback of this method is spurse growth of fastidious microorganisms and some supplemented ingredients of the CAS agar may exert innate antibacterial activity (Perez-Miranda, 2007). Incase chemical tests for siderophores are negative, bioassays can be used for confirmed validation (Merja Manninen and Tiina Mattila-Sandholm, 1994). However, these methods are laborious and inefficient. Hence, necessitate a newer, simple and easy approach to detect microbial siderophore.
A patent search reveals very few patents on the subject. For example, US application 20080193373 describes methods for detecting a microorganism or cell in a subject and methods for detecting, imaging or diagnosing a site, disease, disorder or condition in a subject using microorganisms or cells. US application
20050026140 describes a method for the binding of pathogenic microorganisms and their toxic proteins with ligands that have been covalently tethered at some distance from the surface of a substrate. Indian patent number 190786 describes a process for the preparation of a siderophore belonging to keto bidenate compounds having a nontoxic insecticidal and fungicidial activites
OBJECTS OF THE INVENTION
Accordingly, the main object of the present invention is to provide an easy, sensitive, reproducible, robust and simple but pragmatic approach for detection of microbial siderophore, and process for preparation of dye impregnated strip.
Another objective is to produce the different types of siderophores from microbial source.
The last objective is to evaluate the prepared dye impregnated paper strip for selective application in detection of microbial siderophores.
These objectives are discussed in detail in the following description of the invention and subsequently for the claims.
SUMMARY OF THE INVENTION
To meet the above objects and to overcome the problems existing in the prior art, the present invention describes a new product (dye paper strips) for the identification of microbial siderophore. In accordance with the present invention, a dye-impregnated paper can be used as an indicator dye. In accordance with a more limited aspect of the invention, a dye strip to detect microbial siderophore compromises a filterpaper impregnated with indicator dye. The dye impregnated paper is in a dry form.
The present invention describes the application procedure of developed strip for change in color pattern and its assessment performed by applying a drop of siderophore containing medium.
Different parameters viz. crude culture broth, eluted solvent fraction and purified siderophore activity toward the dye strip has been analyzed. In addition, various factors viz. pH, solvents, organic and inorganic acids, chemical iron chelators, protein and different metal ion solutions were examined for the suitability of present invention so as to confirm its applicability for qualitative detection of siderophore.
A principle advantage of the invention is that the dry strip impregnated with dye can be used anywhere as handy tool without any laboratory facilities. Moreover, small sample (just drop) is enough to test. Consequently, the resulting dye strip product is neat and simple to use.
To date there is no report available on development of dye strip for detection of microbial siderophore. Based on marked color change constitute the main feature of developed strip.
DETAILED DESCRIPTION OF THE INVENTION
An object of the present invention is to provide a process for easy detection of siderophore.
One more object of the present invention to provide a solid support impregnated with detecting material for easy detection of siderophores
It is yet another object of the present invention to reduce use of costly chemicals and laborious handling of solution.
DETAILED DESCRIPTION WITH RESPECT TO EXAMPLES
The following examples are for the purpose of illustration of the present invention and should not be construed to limit the scope of the present invention.
Preparation of dye impregnated strip
1 cm X 5 cm dimension, Whatman Filter No.1 filter paper strips were prepared. Each was treated with freshly prepared 0.5 ml Chrome Azurol Sulphonate (CAS) solution, a siderophore detecting indicator dye, for 4 hours in dark condition and air dried overnight at room temperature. Blue colored appearance of dry strip indicates a correct preparation. These strips were kept in sealed dark carter so as to avoid moisture problem and photo-deactivation of dye impregnated on the strips.
Utilization of dye impregnated strip for siderophore detection
A drop of test solution or broth culture was applied as spot on dye impregnated strip and observed for change in color of spot. The strip was then observed for a minute at room temperature. The brown yellow color against original blue color of strip indicates positive result i.e. presence of the siderophore, whereas no change in coloration of strip indicates negative result or the absence of siderophore. Color change other than brown yellow was recorded as false positive result. Low siderophore concentration may take prolonged period, not more than 5 minutes for color development or till the respective strip get dried at room temperature.
Effect of different pH of deionised water
Different pH values in range of 1 to 14 of deionised water (Millipore system, TDS-0 PPM) were prepared with 6 N HCI and 6 N NaOH. A drop of deionized water of respective pH was applied on dye impregnated strip as test. Water (Milli Q- ultra pure, deionized, 0.22 μ Millipore filtered) set to pH 7 was used as a negative control throughout experimentation.
Effect of different siderophore production media
Different siderophore production media commonly employed were prepared like (a) King's B (consisting of g/L: Glycerine, 10 ; Proteose-peptone, 20; and MgSO4, 1.5), (b) Asparagine medium (consisting of g/L: Asparagine, 5; MgS04, 0.1; and K2HPO4, 0.5), (c) Glycerol medium (consisting of g/L: Glycerol, 10; (NH4)2SO4, 1; MgSO4.7H2O, 1; and
K2HPO4, 4 ), (d) Succinate medium (consisting of g/L: KH2PO4, 6; K2HPO4, 3; (NH4)2SO4, 1; MgSO4.7H2O, 0.2 and sodium succinate, 4), (e) Glucose medium (consisting of g/L: K2HPO4, 0.56; Glucose, 10; Urea, 0.85 and Glutamic acid, 1), (f) Glutamate medium (consisting of g/L: (NH4)2 NO3,1; Na2SO4, 0.02 and NaCI, 0.02), with pH of medium was set to on 7 ±0.1. A drop of prepared media was applied over dye impregnated strip.
Effect of different pH of siderophore production medium
Succinate media most commonly used for siderophore production and its pH was set to pH 3, 5, 7, 9 and 11 with 6N HCI and 6 N NaOH. Succinate medium set to pH 7.0 was treated as negative control.
Effect of different siderophores and their formulation
Two types of siderophores were mainly produced by microbes i.e. hydroxamate and catecholate viz. Pseudomonas putida, hydroxamate producer and Bacillus subtilis, catecholate producer, were obtained from the depository of School of Life Sciences, North Maharashtra University, Jalgaon. Both organisms were confirmed with 16s rRNA at National centre for Cell Science (NCCS), Pune, India. Fermentative production of siderophores by these microbes was carried out in succinate medium (pH 7) for 48 h at 28°C and 180 rpm. Further siderophore was purified through ion exchange chromatography and solvent extraction. Final purification of siderophore was monitored with spectral and HPLC analysis. The culture supernatant as well as solvent elutes along with extracts and purified fractions were checked for the presence of siderophore using dye impregnated strip. About 10 μg/ml purified siderophore obtained from culture broth of each microbe in water (Milli Q- 0.2 μ ultra pure deionized) and crude culture (10 ug/ml) of each microbial growth and solvent elute were examined for test. Midi Q- 0,2 μ ultra pure deionized water set to pH-7 was used as negative control.
Effect of different pH of siderophore solution
Purified siderophore (10 μg/ml) as well as culture supernatant (10 μg/ml) containing hydroxamate type of siderophore from Pseudomonas putida produced in succinate media were set to various pH 3, 7, 9 with 6 N HCI and 6 N NaOH and used as a test
parameter. Ultrapure Milli-Q deionized water set at respective pH was treated as negative control and siderophore adjusted to pH 7.0 was used as reference.
Effect of different pH buffer
Various buffers with wide range of pH were prepared for evaluating their effect on color change of paper strip. For this, Citrate buffer (pH 3, 4, 5), Phosphate buffer (pH 7, 8) and Glycine buffer (pH 9, 10) were examined for their activity against dye impregnated strip. Ultra pure Milli-Q deionized water (pH 7) and hydroxamate type of siderophore (10 ug/ml) were kept as negative and positive control, respectively.
Effect of different eluent and extractant of siderohpore concentration
For siderophore purification, eluent (mostly methanol and ethanol) and extractant (ethyl acetate, chloroform) were treated as test. Siderophore elute fraction in methanol (10 μg/ml hydroxamate siderophore) and extract fraction (10 μg/ml catecholate siderophore) were taken as positive control.
Effect of different alcohol derivatives
Different alcohol derivatives like amyl alcohol, propyl alcohol, benzyl alcohol, butyl alcohol were also examined for its activity against dye impregnated strip. Deionized water (pH 7) and hydroxamate type of siderophore (10 μg/ ml) were kept as negative and positive control, respectively.
Effect of different organic acids:
Various organic acids like L- ascorbic acid, citric acid, succinic acid, boric acid, formic acid, acetic acid, perchloric acid and propionic acid of 1 % concentration were checked for effect on dye strip. Milli-Q ultra pure, deionized water (pH 7) and hydroxamate type of siderophore (10μg/ ml) were kept as negative and positive control, respectively.
Effect of different protein content in sample
For checking interference of proteins on dye strips, different protein samples like casein, bovine serum albumin, peptone, globuline protein each of 1% concentration were tested against Milli-Q ultra pure, deionized water (pH 7) and hydroxamate type of siderophore (10μg/ ml) which served as negative and positive control, respectively.
Effect of different metal ion solutions
Different mono- and di-valent metal ion solution viz. NaCI, MgSO4 7H2O, ZnCI2, CaCI2, CuSO4 and FeSO4 were prepared to 1rnM concentration. Each metal ion solution in production siderophore medium (succinate medium) was examined for their activity on dye. impregnated strip. Deionised Milli-Q ultra pure, deionized water (pH 7) and hydroxamate type of siderophore (10 μg/ ml) served as negative and positive control, respectively.
Effect of different chemical iron chelators:
Various competitive iron chelators like 8-Hydroxy quinoline (10μg/ ml), EDTA (10 μg/ ml) and Catechol (10 μg/ ml) on dye impregnated strips were examined. Milli-Q ultra pure, deionized water (pH 7) and hydroxamate type of siderophore (10 μg/ mi) served as negative and positive control, respectively for its selectivity toward siderophore.
Comparative spectroscopic measurement of siderophore, media and buffer with
Equal volume of CAS-deionised water reaction, CAS-siderophore reaction, CAS-medium (succinate medium) reaction and CAS- buffer (citrate buffer pH-5) carried out at room temperature for spectrum analysis on UV- visible spectrophotometer. The results of examples 1 to 15 are described jointly in Table 1
Table 1: Evaluation of dye strip for its applicability toward siderophore at different parameters
1. A method for detection of siderophore comprising:
a. Impregnating the said dye on a solid support
b. Incubating the solid support and dye for a fixed period
c. Preserving the dye impregnated solid support under light limiting
2. A method for detection of siderophore as in claim 1, wherein, the solid support is absorbent filter paper
3. A method for detection of siderophore as in claims 1 and 2, wherein the dye is CAS reagent
4. A method for detection of siderophore as in claims 1 and 3, wherein, the solid support impregnated with the dye is dried at room temperature for overnight in dark
5. A method for detection of sfderophore as in claims 1, and 4, wherein the incubation may be carried for a maximum period of 24 hours
6. An assay strip for the detection of siderophore
7. An assay strip for detection of siderophore and a method of detection using the same, substantially as described herein and illustrated with the help of examples
• Mr. Kadam Meghraj Shaligram
• Dr. Amoral Babulal Chaudhari
• Prof. Sudhir Bhaskarrao Chincholkar
|Indian Patent Application Number||31/MUM/2010|
|PG Journal Number||28/2013|
|Date of Filing||05-Jan-2010|
|Name of Patentee||KADAM MEGHRAJ SHALIGRAM|
|Applicant Address||DEPT.OF MICROBIOLOGY,SCHOOL OF LIFE SCIENCES, NORTH MAHARASHTRA UNIVERSITY,POST BOX NO.80, JALGAON-425001(MS) INDIA|
|PCT International Classification Number||C12Q1/04|
|PCT International Application Number||N/A|
|PCT International Filing date|