Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2002-03-22
2004-12-21
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S029000, C435S136000, C435S253100, C435S975000, C436S071000, C532S001000
Reexamination Certificate
active
06833249
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a rapid, sensitive, simple, and cost-effective spectrophotometric method of detecting and quantifying mycolic acid in a mycolic acid-fuschin dye complex with absorbance maxima ranging between 490-500 nm in the presence of various test compounds, for screening mycolic acid biosynthesis inhibitors useful as anti-microbial agents and a diagnostic kit thereof comprising basic fuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml, phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1 (v/v), and phenol and distilled water in the ratio ranging between 1:14 to 1:25.
BACKGROUND OF THE INVENTION
Tuberculosis caused by
Mycobacterium tuberculosis
is a public health problem, which has increased in importance during the last two decades, due in part to the increasing number of cases caused by the association of acquired immunodeficiency syndrome (AIDS) and the appearance of multiple drug-resistant strains. Other mycobacteria which are often indistinguishable from tuberculosis have also increased.
The current method of detection is by determining the mycolic acid patterns from clinical isolates of sputum, cerebrospinal fluid, bronchial washing, corneal ulcer, and bone marrow, as well as from acid-fast stain smear-positive clinical specimens. Standardized mycolic acid extraction methods are used to ensure the maximal extraction of mycolic acid derivatives to enhance the sensitivity of the method. Different chromatographic columns are used to identify the species of mycolic acid. The immediate detection of bacteria containing mycolic acid is through acid fast staining and detection through the microscope. Lipid-rich cell walls are not permeable by ordinary stains.
The stain consisting of a basic dye (fuchsin) and phenol (a lipid solvent) is used for the purpose. Phenol partially solubilizes the cell wall and allows fuchsin to penetrate the wall and bind to mycolic acid.
After fuchsin is incorporated, it is resistant to decolorization even after exposure to acid alcohol, a property that characterizes mycobacteria and that can be performed on unprocessed clinical specimens, concentrated specimens, or cultures.
This principle is widely used to identify bacteria producing mycolic acid with the help of a microscope. But an improved system based on visual detection of the mycolic acid is the need of the hour to screen out potential mycolic acid biosynthesis inhibiting compounds from a vast array of plant compounds.
The detection and quantification procedure can be made more accurate by using the help of spectrophotometer. The amount of inhibition can be quantified simply through optical density measurement at a particular wavelength. This does not necessitate the complex, high cost technologies like IR, NMR, GC spectroscopy for the detection, which are currently being used.
The amount of mycolic acid which cannot be quantified through acid fast staining followed by microscopic detection can be quantified by systematic extraction and dye binding followed by spectrophotometric analysis. Keeping this in mind, a simple, rapid, cost effective procedure kit was devised to detect and quantify the mycolic acid which in turn resulted in a high efficiency screen to identify potential inhibitors of mycolic acid biosynthesis.
The protocols for extraction, isolation of mycolic acid and detection of bacteria-producing mycolic acid through acid fast staining are known in the art. But the system as a whole is inefficient and requires a microscope to detect the presence of the bacteria-producing mycolic acid.
Similarly, extraction, isolation and characterization of mycolic acid is carried out through different methods of chromatography and IR, NMR spectroscopy. The emphasis of the present invention was to generate a system to screen a large amount of plant extracts and compounds with potential mycolic acid biosynthesis inhibiting activity, rapidly and with a low cost.
OBJECTS OF THE INVENTION
The main object of the present invention is to develop a quick, simple, sensitive, cost-effective method of identifying mycolic acid.
Another main object of the present invention is to develop a quick, simple, sensitive, cost-effective method of quantifying mycolic acid.
A further object of the present invention is to develop a quick, simple, sensitive, cost-effective spectrophotometric method of identifying and quantifying mycolic acid.
Yet another object of the present invention is to use carbol-fuschin dye and mycolic acid complex to develop a quick, simple, sensitive, cost-effective spectrophotometric method of identifying and quantifying mycolic acid.
Still another object of the present invention is to develop a method of screening test compounds for their mycolic acid inhibitory properties.
Still another object of the present invention is to develop a diagnostic kit for the detection of mycolic acid by spectrophotometry.
SUMMARY OF THE INVENTION
The present invention relates to a rapid, sensitive, simple, and cost-effective spectrophotometric method of detecting and quantifying mycolic acid in a mycolic acid-fuschin dye complex with absorbance maxima ranging between 490-500 nm in the presence of various test compounds, for screening mycolic acid biosynthesis inhibitors useful as anti-microbial agents and a diagnostic kit thereof comprising basic fuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml, phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1 (v/v), and phenol and distilled water in the ratio ranging between 1:14 to 1:25.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention relates to a rapid, sensitive, simple, and cost-effective spectrophotometric method of detecting and quantifying mycolic acid in a mycolic acid-fuschin dye complex with absorbance maxima ranging between 490-500 nm in the presence of various test compounds, for screening mycolic acid biosynthesis inhibitors useful as anti-microbial agents and a diagnostic kit thereof comprising basic fuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml, phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1 (v/v), and phenol and distilled water in the ratio ranging between 1:14 to 1:25.
According to an embodiment of the present invention there is provided a rapid, simple, sensitive, and cost effective method of screening mycolic acid biosynthesis inhibitors useful as anti-microbial agents by quantifying the amount of mycolic acid produced by bacteria in the presence and absence of test compound or extract.
According to another embodiment of the present invention, there is provided the step growing separate cultures of the specified bacteria in the presence and absence of the specified compound or extract for time duration ranging between 40-60 hours.
According to yet another embodiment of the present invention, a lyophilizing bacterial pellet is produced.
According to still another embodiment of the present invention, there is provided the step of extracting mycolic acid by a conventional method.
According to still another embodiment of the present invention, there is provided the step of dissolving extracted mycolic acid extract in hexane to obtain mycolic acid solution.
According to still another embodiment of the present invention, there is provided the step of adding the specified solution to carbol-fuschin dye in the ratio ranging between 1:4 to 4:1 (v/v).
According to still another embodiment of the present invention, there is provided the step of shaking the product of the above step vigorously to obtain a pink color mycolic acid-dye complex as an upper layer.
According to still another embodiment of the present invention, there is provided the step of quantifying the specified mycolic acid spectrophotometrically at a wavelength ranging between 490-500 nm.
According to still another embodiment of the present invention, there is provided the step of determining the degree of inhibition of mycolic acid biosynthesis in the compound-treated bacterial culture.
In still another embodiment of the present invention, wh
Khanuja Suman Preet Singh
Kumar Tiruppadiripuliyur Ranganathan Santha
Shasany Ajit Kumar
Srivastava Suchi
Council of Scientific and Industrial Research
Dickinson Wright PLLC
Hanley Susan
Witz Jean C.
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