KR20050068350A - Rapid method for measurement of microbiol - Google Patents
Rapid method for measurement of microbiol Download PDFInfo
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- KR20050068350A KR20050068350A KR1020030099640A KR20030099640A KR20050068350A KR 20050068350 A KR20050068350 A KR 20050068350A KR 1020030099640 A KR1020030099640 A KR 1020030099640A KR 20030099640 A KR20030099640 A KR 20030099640A KR 20050068350 A KR20050068350 A KR 20050068350A
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Abstract
본 발명은 미생물의 신속한 측정법에 관한 것으로, 미생물의 ATPase 활성 측정법 및 미생물의 ATPase 활성 측정에 필요한 시약을 개발하고, 표준한천평판법에 의한 미생물의 총균수와 미생물의 ATPase 활성과의 관계로부터 소프트웨어를 개발함으로써, 대략 1시간 이내에 10 검체 이상의 미생물 총균수를 현장에서도 즉시 신속하게 측정할 수 있어, 실험실은 물론 원하는 현장에서 각종 식품에 대한 미생물의 검사를 시간과 장소에 구애받지 않고 다목적으로 실시할 수 있다.The present invention relates to a rapid measurement method of microorganisms, to develop reagents for measuring ATPase activity of microorganisms and ATPase activity of microorganisms, and to develop software from the relationship between the total microbial count of microorganisms and ATPase activity of microorganisms by standard agar plate method. Thus, the total number of microorganisms of 10 or more specimens can be quickly and quickly measured in the field within approximately one hour, and the microorganisms can be tested for various foods at various places regardless of time and place at the laboratory as well as the desired site. .
Description
본 발명은 미생물의 신속한 측정법에 관한 것으로, 미생물의 ATPase 활성 측정법 및 미생물의 ATPase 활성 측정에 필요한 시약을 개발하고, 표준한천평판법에 의한 미생물의 총균수와 미생물의 ATPase 활성과의 관계로부터 소프트웨어를 개발함으로써, 대략 1시간 이내에 10 검체 이상의 미생물 총균수를 현장에서도 즉시 신속하게 측정할 수 있어, 실험실은 물론 원하는 현장에서 각종 식품에 대한 미생물의 검사를 시간과 장소에 구애받지 않고 다목적으로 실시할 수 있다.The present invention relates to a rapid measurement method of microorganisms, to develop reagents for measuring ATPase activity of microorganisms and ATPase activity of microorganisms, and to develop software from the relationship between the total microbial count of microorganisms and ATPase activity of microorganisms by standard agar plate method. Thus, the total number of microorganisms of 10 or more specimens can be quickly and quickly measured in the field within approximately one hour, and the microorganisms can be tested for various foods at various places regardless of time and place at the laboratory as well as the desired site. .
산업이 발달하고 생활이 윤택해지면서 다양한 식문화가 전개되고 있고, 이와 더불어 식품위생의 중요성도 더욱 부각되고 있다.As the industry develops and the life is improved, various food cultures are being developed, and the importance of food hygiene is increasing.
최근 식품과 관련하여 식중독 사고가 매년 증가하고 있는 실정이나, 이에 대한 대책은 매우 미흡한 점이 사실이다. In recent years, food poisoning accidents are increasing year by year, but the countermeasures are insufficient.
이러한 이유는, 사전에 식중독을 유발시키는 요인을 제거하지 못하기 때문인 것으로 사료되며, 특히 미생물에 대해서 신속하게 검사할 수 있는 방법이 거의 없기 때문인 것으로 사료된다. This reason is considered to be because the factor causing food poisoning cannot be eliminated in advance, and in particular, there is almost no method for rapid testing for microorganisms.
종래의 미생물 균수의 측정방법은 배지(표준 한천 평판법)를 제조하여 미생물을 접종한 후 3∼4일간 배양하여 미생물 균수를 분석하는 것이 일반적이다. In the conventional method for measuring the number of microbial bacteria, it is common to prepare a medium (standard agar plate method), inoculate microorganisms, and incubate for 3 to 4 days to analyze the microbial bacteria.
최근에는 보다 신속한 측정법이 개발되기 시작하였는데, 예컨대, 3M Patrifilm에 의한 분석법, ATP Bioluminascence assay, DNA Probe를 이용한 PCR법, ELISA 등을 이용한 효소면역학적인 방법 등이 있다.Recently, more rapid measurement methods have been developed, for example, 3M Patrifilm, ATP Bioluminascence assay, PCR using DNA Probe, and enzyme immunoassay using ELISA.
상기 3M Patrifilm에 의한 분석법은, 기존의 배지를 개량하여 측정하기에 편리하도록 제조한 것이다. 이러한 방법은 배지를 만들지 않아도 된다는 장점은 있으나, 배양하는데 많은 시간(2일 이상)이 소요되며, 가격이 대체적으로 고가이다.The assay by 3M Patrifilm is prepared to improve the existing medium and to be convenient for measurement. This method has the advantage of not having to make a medium, but it takes a lot of time (more than 2 days) to cultivate and is generally expensive.
또한, ATP Bioluminascence assay의 경우, 일반적으로 세포는 대사작용에 이용되는 에너지의 근원인 ATP를 가지고 있으며, 미생물의 수가 많으면 이에 따라 상대적으로 ATP 함량도 증가하므로, ATP 함량을 측정함으로서 미생물의 수를 측정할 수 있는 원리를 이용한 것이다. 이 분석법은 비교적 신속하게 측정할 수 있으나, 반응시약이 고가이어서 대부분 기존의 표준 한천 평판법을 활용하고 있다. In addition, in the case of the ATP Bioluminascence Assay, cells generally have ATP, a source of energy used for metabolism, and if the number of microorganisms is large, the ATP content is relatively increased accordingly, and thus the number of microorganisms is measured by measuring the ATP content. It uses the principle that can be done. This method can be measured relatively quickly, but the reaction reagents are expensive and most of them use the standard agar plate method.
상기 DNA Probe를 이용한 PCR법은, DNA를 시험관내에서 대량으로 신속하게 증폭하는 방법을 이용한 것이다. PCR의 구성요소는 Primer set (1, 2), dNTPs, Taq polymerase로 되어있으며, 측정원리는 Primer1 →Primer2 →dNTPs →Taq polymerase Denaturation →Annealing →Extension의 순으로 되어 있다. 그러나 이 측정방법은 균을 동정하는 데에는 신속(24∼30시간)하게 처리할 수는 있으나, 균수를 측정하기에는 어려움이 있다. The PCR method using the DNA probe is a method of rapidly amplifying a large amount of DNA in vitro. PCR components consist of Primer set (1, 2), dNTPs, Taq polymerase, and the measuring principle is Primer1 → Primer2 → dNTPs → Taq polymerase Denaturation → Annealing → Extension. However, this measurement method can be quickly (24-30 hours) to identify the bacteria, but difficult to determine the number of bacteria.
또한, ELISA 등을 이용한 효소면역학적인 방법은, 대상균이 가지고 있는 특이적인 항원에 대하여 인위적으로 제조된 항체(균체)를 결합시키고, 이러한 결합체에 효소 표식인자 및 기질을 반응시켜, 측정기구(ELISA) 등을 이용하여 측정하는 검사법이다. 그러나, 이들은 주로 미생물을 동정하는데 이용되고 있으며, 균수를 측정하는데는 부적합하다.In addition, an enzyme immunological method using an ELISA or the like binds an antibody (bacteria) prepared artificially to a specific antigen possessed by a target bacterium, and reacts an enzyme marker and a substrate with such a conjugate to measure a measuring instrument (ELISA). It is an inspection method to measure using However, they are mainly used to identify microorganisms and are inadequate for measuring the number of bacteria.
따라서, 본 발명에서는 상기 종래의 문제점을 개선한 것으로, 미생물의 ATPase 활성 측정법 및 미생물의 ATPase 활성 측정에 필요한 시약을 개발하고, 표준한천평판법에 의한 미생물의 총균수와 미생물의 ATPase 활성과의 관계로부터 소프트웨어를 개발함으로써, 대략 1시간 이내에 10 검체 이상의 미생물 총균수를 현장에서도 즉시 신속하게 측정할 수 있어, 실험실은 물론 원하는 현장에서 각종 식품에 대한 미생물의 검사를 시간과 장소에 구애받지 않고 다목적으로 실시할 수 있는 미생물의 신속한 측정법을 제공하고자 한다.Therefore, the present invention improves the above-mentioned conventional problems, and develops a reagent for measuring the ATPase activity of the microorganism and the ATPase activity of the microorganism, and from the relationship between the total bacterial count of the microorganism by the standard agar plate method and the ATPase activity of the microorganism. By developing the software, the total number of microorganisms of more than 10 samples can be measured quickly and promptly in the field within about one hour, so that the inspection of microorganisms on various foods in the laboratory and at the desired site can be performed at any time and place. It is intended to provide a rapid measurement method of microorganisms.
이하, 본 발명의 구성에 대해 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated.
본 발명의 미생물의 신속한 측정법은 하기 3단계의 시험 및 개발을 통하여 완성하였다.Rapid measurement of the microorganism of the present invention was completed through the following three steps of testing and development.
1) 미생물의 ATPase 활성 측정법의 개발1) Development of ATPase Activity Measurement Method of Microorganisms
2) 미생물의 ATPase 활성 측정에 필요한 시약의 개발2) Development of reagents for measuring ATPase activity of microorganisms
3) 표준한천평판법에 의한 미생물의 총균수와 미생물의 ATPase 활성과의 관계로부터 Soft Ware의 개발3) Development of Soft Ware from the relationship between total microbial count of microorganisms and ATPase activity of microorganisms by standard agar plate method
여기서, 본 발명의 측정방법은 2가지( I, II )의 시약으로 나뉘어져 있으며, 시약 I은 반응, 시약 II은 발색시키는 작용을 한다. 또한 시약 I은 매우 불안정하여 안정화시켰다.Here, the measuring method of the present invention is divided into two reagents (I, II), and reagent I functions to react and reagent II to develop color. Reagent I was also very unstable and stabilized.
ATPase 활성은 Ca, Mg, EDTA를 포함하는 활성법으로 측정할 수 있는 방법을 개발하였으며, 적당량의 샘플을 시약 I에 첨가한 후 5∼20분간 반응시킨다. 그런 다음, 정지시약을 첨가하여 반응을 정지시킨 후, 시약 II를 첨가하여 10∼20분간 발색시켜 400∼660nm에서의 흡광도를 측정하면 완료된다. ATPase activity was developed by an activity method including Ca, Mg, and EDTA, and an appropriate amount of sample was added to Reagent I and then reacted for 5 to 20 minutes. Then, the reaction is stopped by the addition of a stop reagent, followed by the addition of reagent II to develop color for 10 to 20 minutes to measure the absorbance at 400 to 660 nm.
또한 흡광도는 Potable Spectrophotometer(PSP)를 이용하였으며 PSP 내부에 Soft Ware를 장착하여 흡광도를 측정함으로서 자동적으로 미생물 균수를 알 수 있도록 개발하였다. In addition, the absorbance was measured by using a Potable Spectrophotometer (PSP), and the Soft Ware was installed inside the PSP to measure the absorbance.
이하, 실시예를 통하여 좀더 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
실시예 1(미생물의 ATPase 활성 측정법 개발)Example 1 (Development of a method for measuring ATPase activity of microorganisms)
미생물의 ATPase 활성 측정법은 다음과 같다. Microbial ATPase activity measurement method is as follows.
미생물의 채취는 일반적인 방법과 같은 방법으로 채취한다. 즉, 시료 표면의 미생물을 면봉으로 채취하는 방법과 시료의 적당량을 분리하여(중량을 정확히 측정) 살균수에 첨가하여 현탁시킨다.The microorganisms are collected in the same way as usual. That is, a method of collecting microorganisms on the surface of the sample with a cotton swab and an appropriate amount of the sample is separated (weighed accurately) and added to the sterilized water and suspended.
상기 현탁된 시료를 반응액A에 첨가하여 5∼20분간 반응시킨 후, 정지액(TCA)을 첨가하여 반응을 정지시킨다.The suspended sample is added to the reaction solution A and allowed to react for 5 to 20 minutes, and then the stop solution (TCA) is added to stop the reaction.
이것을 여과한 후 반응액B에 첨가하여 약 20분간 반응시킨 다음, Potable Spectrophotometer(PSP)로 O.D값을 측정하면, PSP안에 장착되어 있는 Soft ware에 의해서 자동적으로 총균수가 측정된다. 이 때에 소요되는 시간은 약 45분 정도이며, 연속적으로 측정할 수 있는 형식이므로, 숙련도에 따라서 1시간에 10∼20 샘플을 측정할 수 있다.After filtering this, the reaction solution B was added and reacted for about 20 minutes, and then the O.D value was measured with a Potable Spectrophotometer (PSP). The total bacterial count was automatically measured by the software installed in the PSP. The time required at this time is about 45 minutes and can be measured continuously. Therefore, 10 to 20 samples can be measured in one hour depending on skill.
상기 본 발명의 미생물의 ATPase 활성 측정법을 요약하면 하기 표1과 같다.A summary of the ATPase activity measurement method of the microorganism of the present invention is shown in Table 1 below.
실시예 2(미생물의 ATPase 활성 측정에 필요한 시약 개발)Example 2 (Development of Reagents for Measuring ATPase Activity of Microorganisms)
반응시약은 크게 2가지로 나눌 수 있으며, 그 중 가장 핵심이 되는 반응시약은 ATP를 포함하는 반응시약으로써, 현장에서 측정할 수 있도록 ATP 농도를 1∼20mM로 조절하였으며, 그 외에, ATP 농도에 맞추어 칼슘, 마그네슘, EDTA 농도 등을 1∼10mM로 조절하였다. The reaction reagent can be largely divided into two types, and the most important one is the reaction reagent containing ATP, and the ATP concentration is adjusted to 1-20 mM so that it can be measured in the field. The calcium, magnesium, and EDTA concentrations were adjusted to 1-10 mM.
또한, 여기에 반응시약을 안정화시키기 위하여, 반응시약을 물리적(초고압, 통전가열 및 고압열처리)으로 처리하였다.In addition, in order to stabilize the reaction reagents, the reaction reagents were treated physically (ultra high pressure, energized heating and high pressure heat treatment).
도1은 본 발명의 방법으로 처리한 시약 A의 안정성을 나타낸 그래프이다.1 is a graph showing the stability of Reagent A treated by the method of the present invention.
도면에서와 같이, 본 발명의 방법으로 처리한 반응시약은 약 100일간 실내에서 저장하여도 안정하였다.As shown in the figure, the reaction reagent treated by the method of the present invention was stable even when stored indoors for about 100 days.
실시예 3(표준한천평판법에 의한 미생물의 총균수와 미생물의 ATPase 활성과의 관계로부터 soft ware의 개발)Example 3 (Development of software from the relationship between total microbial count of microorganisms and ATPase activity of microorganisms by standard agar plate method)
표준한천평판법에 의한 미생물의 총균수와 미생물의 ATPase 활성과의 관계로부터, ATPase 활성이 미생물의 총균수를 측정할 수 있는지에 대한 검증과 양자간의 관계로부터 산출한 관계식을 이용하여 Soft Ware를 개발하였다.From the relationship between the total microbial count of microorganisms and the ATPase activity of microorganisms by standard agar plate method, we verified whether ATPase activity can measure the total microbial count of microorganisms and developed the soft ware using the relational equation calculated from the relationship between them. .
한천평판법에 의한 미생물의 총균수와 ATPase 활성에 의한 미생물의 총균수와의 관계는 2단계의 직선관계를 나타내었으며, 105까지는 천천히 105이상에서는 급격히 상승하는 경향을 나타내었다.The relationship between the total cells of the microorganism according to the total number of bacteria and the ATPase activity of the microorganism according to the agar pyeongpanbeop has exhibited a linear relationship between the step, through 10 5, 10 5 or more slowly tended to rapidly rise.
따라서 2단계의 직선관계로부터 각각의 관계식을 산출하면, 초기의 1단계에서는 y = 0.0053x + 0.1254의 식이 성립되었으며, 2단계에서는 y = 0.029x - 0.0027의 관계식이 성립되었다.Therefore, when each relation was calculated from the linear relations of two stages, the equation of y = 0.0053x + 0.1254 was established in the first stage, and the relation of y = 0.029x-0.0027 was established in the second stage.
도2는 표준한천평판법에 의한 미생물의 총균수와 미생물의 ATPase 활성과의 관계를 나타내는 그래프이다.Figure 2 is a graph showing the relationship between the total microbial count of microorganisms and ATPase activity of microorganisms by the standard agar plate method.
이상의 결과로부터, ATPase 활성 측정법을 이용하면 미생물의 총균수를 측정할 수 있었으며, 각각의 식을 이용하여 Soft Ware를 개발한 결과, ATPase 활성법에 의한 미생물의 총균수를 신속하고 정확하게 측정할 수 있었다.Based on the above results, the ATPase activity assay was used to measure the total microbial count of microorganisms. As a result of developing Soft Ware using the respective formulas, the total microbial count of microorganisms by the ATPase activity method was able to be measured quickly and accurately. .
이상 설명한 바와 같이, 본 발명에 따르면, 미생물의 ATPase 활성 측정법 및 미생물의 ATPase 활성 측정에 필요한 시약을 개발하고, 표준한천평판법에 의한 미생물의 총균수와 미생물의 ATPase 활성과의 관계로부터 소프트웨어를 개발함으로써, 대략 1시간 이내에 10 검체 이상의 미생물 총균수를 현장에서도 즉시 신속하게 측정할 수 있어, 실험실은 물론 원하는 현장에서 각종 식품에 대한 미생물의 검사를 시간과 장소에 구애받지 않고 다목적으로 실시할 수 있어, 관련 분야에의 이용 및 응용이 기대된다 하겠다. As described above, according to the present invention, by developing a reagent for measuring the ATPase activity of the microorganism and ATPase activity of the microorganism, by developing software from the relationship between the total microbial count of the microorganism and the ATPase activity of the microorganism by the standard agar plate method The total number of microorganisms more than 10 specimens can be measured promptly at the site within about an hour, and the microorganisms of various foods can be inspected at any place, as well as in the laboratory, regardless of time and place. It is expected to be used and applied in related fields.
도1은 본 발명의 방법으로 처리한 시약 A의 안정성을 나타낸 그래프1 is a graph showing the stability of Reagent A treated by the method of the present invention.
도2는 표준한천평판법에 의한 미생물의 총균수와 미생물의 ATPase 활성과의 관계를 나타내는 그래프이다.Figure 2 is a graph showing the relationship between the total microbial count of microorganisms and ATPase activity of microorganisms by the standard agar plate method.
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