KR20140081335A - Screening method for cattle tuberculosis by using electronic nose - Google Patents

Abstract

The present invention relates to a method for diagnosing bovine tuberculosis in cattle, and more particularly, to a method for screening for bovine tuberculosis using an electronic nose equipped with a metal oxide sensor (MOS).
According to the present invention as described above, it is possible to automatically screen a small tuberculosis infection sera and an uninfected serum with an electronic nose system through a single sample injection process, thereby rapidly screening suspicious objects of a small number of small tuberculosis infections, There is a saving effect.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a screening method for bovine tuberculosis using an electronic nose,

The present invention relates to a method for diagnosing bovine tuberculosis in cattle, and more particularly, to a method for screening for bovine tuberculosis using an electronic nose equipped with a metal oxide sensor (MOS).

In the country, bovine tuberculosis managed by the Livestock Infectious Diseases Prevention Act is the most effective anti-tuberculosis policy, and the bovine population is being disposed of and compensation is paid (livestock epidemic prevention law (Law 10930, July 25, 2011)). In particular, since bovine tuberculosis is a common infectious disease, rapid and precise diagnosis of bovine tuberculosis is very important for public health.

Various pathological, bacteriological, genetic, cytomechanical, and serological diagnostic methods have been developed by many researchers in the past, and patent publications include Korea Patent Publication No. 10-2011-0052295 Diagnostic solution and method for diagnosing small tuberculosis using the same) Korean Patent Laid-Open Publication No. 10-2012-0051196 (Nuclear Diagnostic Kit for Nuclear Medicine and Method for Diagnosing Nuclear Nuclei Using the same), Korean Patent No. 10-0951057 An enzyme immunoassay kit, and a method for diagnosing a nosocomial nucleus using the same).

Pathologic diagnosis of tuberculous nodules and diagnosis of tuberculosis are pathologic diagnosis and identification of bovine tuberculosis in addition to bacteriological diagnosis of tuberculosis. The bacterium Mycobacterium tuberculosis contains more lipid components than other general bacteria, and its generation time is about 24 hours, which is about 20 times longer than general bacteria. Therefore, the bacteriological diagnosis that identifies and identifies small bacterium tuberculosis requires a minimum of eight weeks or more for bacteria identification only. In recent years, a mycobacterial detection system (MGIT ^™ 960 Mycobacterial Detection System) for measuring metabolites of bacterium Mycobacteria has been developed and used for the diagnosis of bovine tuberculosis. However, it is expensive because equipment and consumables are expensive.

There is a polymerase chain reaction (PCR) that amplifies a bovine tuberculosis specific gene region by a genetic diagnosis method. There are lymphoproliferative reaction and interferon method as a cell immunological diagnosis method. Currently, It is used as an official diagnosis method of tuberculosis. Interferon assay is one of the methods of definitive diagnosis of small bovine tuberculosis. The difference of the amount of interferon produced by stimulating the blood of bovine bovine spongiform encephalopathy with M. avium PPD tuberculin and M. bovis PPD tuberculin is measured . If the amount of interferon secreted from bovine tuberculous bacteriophage stimulated blood is greater than the amount of interferon secreted from the tuberculous bacteriophage stimulated blood, it is diagnosed as bovine tuberculosis and lowers the rate of misdiagnosis due to non-specific reaction caused by tuberculosis infection.

In addition, enzyme immunoassay (ELISA) is used as a serological diagnostic method, and it takes about 4 hours for a skilled examiner to treat 100 samples.

In Korea, PPD tuberculin, a protein derived from bacterium Mycobacterium tuberculosis, is inoculated into the anterior muscle wall and measured for skin thickness before and after inoculation 72 hours after the inoculation. If the difference is more than 5 mm, the diagnosis is positive. The intradermal diagnosis method has a limit of two visits and 72 hours of long diagnosis time.

As described above, the conventional diagnostic methods have a disadvantage that they require a lot of labor, a long diagnostic period, expensive equipment, and consumables.

Meanwhile, the electronic nose system, which is used in various industrial fields such as food, environment, beverage, pharmaceutical, packaging, cosmetics, automobile and agriculture, / mass spectrometry) method.

The electronic nose system compiles the raw data of volatile organic compounds (VOC) measured by MOS (metal oxide sensor) to create a VOC library and apply various statistical processing methods to the purpose of the analysis The statistical processing system of the electronic nose system is PCA (Principal Component Analysis), DFA (Discriminant Factorial Analysis), PLS (Partial Least Square), SIMCA (Soft Independent Modeling Class Analogy) (Statistical Quality Control), and SLM (Shelf Life Method).

Among the statistical processing systems, PCA is a classification method based on various parameters of the components representing volatile organic compounds. The method is a method of identifying the difference of volatile organic compounds correlated with each other without a reference sample. As a method to confirm the difference. The DFA is a data application technology that builds a library by repeatedly applying a sample with a certain information to the device and then inserts the analysis result of the unknown sample into the library to confirm unknown sample information. The PLS is a method of analyzing the reference sample by concentration and creating a standard calibration curve to confirm the concentration of the unknown sample. SIMCA is a method that can determine whether the sample is within the allowable range by setting the allowable range using the reference sample and then analyzing the unknown sample. The SQC repeatedly applies the electronic nose system for a certain period of time using the reference sample, and sets the allowable range by the user. After repeated application, unknown sample is analyzed and it can be confirmed that it is normal quality if it is included in the allowable range. SLM is a method to check VOC change with time of sample at a glance, and can confirm the shelf life of the food and the stability of the product according to the storage method.

Microbacterium tuberculosis bacterium is cultivated and emits the unique volatile organic compounds methyl phenylacetate, methyl p-anisate, methyl nicotinate and o-phenylanisole. Using these characteristics, volatile organic compounds in the serum of the nose are analyzed to promptly screen the infected individuals.

Since the microbuclear nuclear screening method using the electronic nose of the present invention can quickly test a large amount of serum, only those that have been identified as positive by primary screening with an electronic nose prior to intradermal testing are subjected to intradermal diagnosis If confirmed, the labor and time for diagnosis of small tuberculosis can be drastically reduced.

An object of the present invention is to provide an electronic nose system to which discriminant analysis and major component analysis are applied to analyze volatile organic compounds in bovine serum and quickly and automatically screen the bovine tuberculosis infected serum and the non-infected serum, And to solve the problems of the small tuberculosis diagnosis method which consumes equipment and consumables.

In order to accomplish the above object, the present invention provides a method for diagnosing bovine tuberculosis of bovine (1), comprising the steps of: (1) separating bovine serum; (2) detecting volatile organic compounds (VOC) in the separated serum using an electronic nose equipped with a metal oxide sensor (MOS); And (3) analyzing the sensed volatile organic compounds through discriminant factor analysis (DFA) or principal component analysis (PCA); The method comprising the steps of:

In the step (2), the volatile organic compound may be at least one selected from the group consisting of fluorine, chlorine, nitrogen monoxide, ozone, ammonia, amines, propane, butane, hydrocarbon, hydrocarbons, methane, solvents, toluene, xylene and alcohol.

In step (2), the metal oxide sensor may include one to three sensors for sensing oxidizing gases, one to three sensors for detecting toxic gases, one for sensing flammable gases, And three or more sensors, and one or three sensors for sensing organic compounds.

According to the present invention as described above, it is possible to automatically screen a small tuberculosis infection sera and an uninfected serum with an electronic nose system through a single sample injection process, thereby rapidly screening suspicious objects of a small number of small tuberculosis infections, There is a saving effect.

In addition, it can help the rapid disinfection of bovine tuberculosis causing huge economic damage to domestic cattle farmers. It can prevent and prevent damage by early diagnosis and early prevention of bovine tuberculosis, and it can be extended to other epidemic screening diagnosis .

FIG. 1 shows the results of discriminating between bovine tuberculosis infection sera and non-infected sera by the discriminant factor analysis according to the present invention.
FIG. 2 shows the result of discriminating between sera from bovine tuberculosis and non-infected sera according to the analysis of main components according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method of diagnosing bovine tuberculosis of bovine (1), comprising the steps of: (1) separating serum from bovine; (2) detecting volatile organic compounds (VOC) in the separated serum using an electronic nose equipped with a metal oxide sensor (MOS); And (3) analyzing the sensed volatile organic compounds through discriminant factor analysis (DFA) or principal component analysis (PCA); The method comprising the steps of:

The metal oxide sensor (MOS) type electronic nose uses a multi-sensor array technology to convert a specific fragrance or odor component into an electrical signal by causing an electrochemical reaction in each sensor. Using pattern recognition software such as imitative discriminant analysis and principal component analysis, it is possible to distinguish odors and distinguish between samples.

More specifically, when oxygen comes into contact with the sensor, electrons are taken away to increase the electrical conductivity. In this case, the presence of a reducing substance in the gas containing the odor component reduces the increase of the electric conductivity.

Therefore, each sensor in the electronic nose system and the statistical processing method suited to the analysis purpose are important factors.

Before applying the method of screening for bovine tuberculosis according to the present invention, the raw data of volatile organic compounds (VOC) measured by a metal oxide sensor (MOS) are synthesized Create a VOC library. The accuracy of discrimination is increased as more data is stored in storing the database of normal serum and infected sera, and the degree of similarity between the target sample and the group to be analyzed is analyzed, Can be distinguished. If it shows a similarity of 70% or more, it is judged to be infected sera but may raise or lower the above criteria.

In the step (2), the volatile organic compound may be at least one selected from the group consisting of fluorine, chlorine, nitrogen monoxide, ozone, ammonia, amines, propane, butane, hydrocarbons, methane, solvents, toluene, xylene, and alcohols.

In step (2), the metal oxide sensor may include one to three sensors for sensing oxidizing gases, one to three sensors for detecting toxic gases, one for sensing flammable gases, And three or more sensors for detecting the organic compounds.

More preferably, two sensors for sensing at least one oxidizing gas selected from the group consisting of fluorine, chlorine, nitrogen monoxide (NO), ozone, ammonia, Two sensors for detecting one or more toxic gases selected from the group comprising amines, at least one selected from the group consisting of propane, butane, hydrocarbons, methane, It is best to use three sensors that detect one or more organic compounds selected from the group consisting of three sensors that detect flammable gases, solvents, toluene, xylene, and alcohol. .

A large amount of bovine serum can be quickly and conveniently distinguished from infected and non-infected sera by detecting volatile organic compounds (VOC) in the serum of a bovine using the metal oxide sensor as described above and applying discriminant analysis or major component analysis .

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1.

11 cows infected with M. bovis and 10 non-sensitized chlorine were reported on the open field farms.

Blood was collected from each cow, serum was then separated, placed in sterile Eppendorf tubes, and stored in a -20 ° C freezer until analysis by electronic nose.

Then, 100 μl of the dissolved serum and 400 μl of a 0.9% (w / v) NaCl aqueous solution of sterilized were mixed well, and the mixture was added to a 10 ml electron It was placed in a headspace vial and immediately sealed with a crimp cap with silicon-Teflon septum. The sealed vials were allowed to react for 45 minutes at 37 < 0 > C and then published in electronic nose analysis.

Volatile organic compounds (VOC) in serum samples obtained from each of the above cows were detected using an FOX 3000 electronic nose of Alpha MOS (Multi Organoleptic Systems) equipped with 12 metal oxide sensors (MOS sensors) 1, and discriminant factorial analysis (DFA). Each serum was tested 3 times repeatedly.

As a result, according to discriminant factor analysis (DFA), the classification power of the horizontal axis DF1 was 100 and the classification power of the vertical axis DF2 was 0. As a result of discriminating power of the horizontal axis, 11 small bovine tuberculosis sera and 10 The bovine tuberculosis-uninfected serum was distinctly differentiated (see Fig. 1).

Example 2.

In the same manner as in Example 1, small blood tuberculosis infection sera and bovine tuberculosis non-infected sera were distinguished from each other and analyzed by principal component analysis (PCA).

Three principal components (PC) were used in this analysis, and indices of PC 1, 2 and 3 were 62.8, 36.1 and 0.9, respectively (see FIG. 2).

Also, as shown in FIGS. 1 and 2, one blue box located apart may be an error due to other environmental factors when injecting the sample, or may be a different third odor, If the group is formed, it is considered that it is not infected or non - infected sera and it is necessary to find other factors that cause change in the smell of serum which may be a new disease.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (3)

In a method for diagnosing bovine tuberculosis,
(1) separating the serum of the bovine;
(2) detecting volatile organic compounds (VOC) in the separated serum using an electronic nose equipped with a metal oxide sensor (MOS); And
(3) analyzing the sensed volatile organic compounds through discriminant factor analysis (DFA) or principal component analysis (PCA); Lt; RTI ID = 0.0 > TB < / RTI >
The method according to claim 1,
In the step (2), the volatile organic compound may be at least one selected from the group consisting of fluorine, chlorine, nitrogen monoxide, ozone, ammonia, amines, propane, butane, hydrocarbons, methane, solvents, toluene, xylene and alcohol. The method for screening for bovine tuberculosis in cattle according to claim 1,
The method according to claim 1,
In step (2), the metal oxide sensor may include one to three sensors for sensing oxidizing gases, one to three sensors for detecting toxic gases, one for sensing flammable gases, And three to three sensors for detecting organic compounds. The method of screening for bovine tuberculosis of cattle according to claim 1,

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