KR101478310B1 - Improved method for detecting Campylobacter using syringe filter - Google Patents

Improved method for detecting Campylobacter using syringe filter Download PDF

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KR101478310B1
KR101478310B1 KR1020130018265A KR20130018265A KR101478310B1 KR 101478310 B1 KR101478310 B1 KR 101478310B1 KR 1020130018265 A KR1020130018265 A KR 1020130018265A KR 20130018265 A KR20130018265 A KR 20130018265A KR 101478310 B1 KR101478310 B1 KR 101478310B1
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syringe filter
campylobacter
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서건호
천정환
김홍석
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건국대학교 산학협력단
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Abstract

본 발명은 실린지 필터법을 이용한 개선된 캠필로박터 검출 방법에 관한 것이다.The present invention relates to an improved method for detecting a camphor bulb using a syringe filter method.

Description

실린지 필터법을 활용한 개선된 캠필로박터 검출 방법{Improved method for detecting Campylobacter using syringe filter}Technical Field [0001] The present invention relates to an improved method for detecting Campylobacter using syringe filter,

본 발명은 실린지 필터법을 이용한 개선된 캠필로박터 검출 방법에 관한 것이다.The present invention relates to an improved method for detecting a camphor bulb using a syringe filter method.

일반적으로 Campylobacter spp.는 그람 음성의 미호기성 세균으로서 인체에 감염되어 식중독을 일으키는 병원성 세균이다. Campylobacter spp.로 인한 식중독 사고는 전세계적으로 발생되고 있으며, 특히 북미와 유럽 등의 선진국에서는 Salmonella에 의한 감염증보다 발생빈도가 많을 정도로 문제가 되고 있다. 가장 빈번하게 식중독을 일으키는 종은 Campylobacter jejuni (C. jejuni)와 Campylobacter coli (C. coli)로서 전체 Campylobacter 감염증의 약 90%이상을 차지한다. Campylobacter spp. Is a gram-negative, aerobic bacterium that is infectious to humans and causes food poisoning. Food poisoning accidents caused by Campylobacter spp. It has been occurring throughout the world, especially in developed countries such as Salmonella in North America and Europe Which is more frequent than infectious diseases caused by infection. Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the most frequent cause of food poisoning, accounting for more than 90% of all Campylobacter infections.

식품 내 C. jejuni/C. coli 검출 시, 국내외 공인검출법에서는 선택배지를 사용하는 배지배양법을 표준화된 검출법으로 사용하고 있다. 증균배지를 사용하여 고온에서 최대 72시간 증균한 후, 다양한 선택배지를 활용하여 집락을 선택적으로 검출한다. 그러나 최근에는 캠필로박터 배지에 함유된 항생제인 cefoperazone에 내성을 보이는 세균이 계육(닭고기)에서 많이 증가하는 추세이다. 이러한 경우 경쟁집락이 증균 및 선택배지에서 높은 수준으로 자라 캠필로박터의 선택적인 배양을 어렵게 한다. 따라서 이러한 균을 적절히 배제시키는 것이 절대적으로 필요하다.
C. jejuni / C. in food . For the detection of E. coli , the culture medium using the selective medium is used as the standardized detection method for both the domestic and the international certified detection methods. After incubation at elevated temperature for up to 72 h using a growth medium, colonies are selectively detected using a variety of selective media. Recently, however, bacteria showing resistance to cefoperazone, an antibiotic contained in Campylobacter media, are increasing in chicken meat. In this case, competing colonies grow to a high level in the enrichment and selection medium, making selective culturing of the Campylobacter difficult. Therefore, it is absolutely necessary to properly exclude these bacteria.

관련 선행특허로 대한민국특허공개번호 제1020130013269호는 캠필로박터에 대한 민감성을 개선한 배지 조성물 및 그 제조방법에 관한 것이고,Korean Patent Publication No. 1020130013269, which is related to the related patent, relates to a culture medium composition improved in sensitivity to a camphyllobum and a preparation method thereof,

다른 관련 선행특허로 대한민국특허공개번호 제1020130013269호는 캠필로박터에 대한 민감성을 개선한 배지 조성물 및 그 제조방법에 관한 것이고,
Korean Patent Publication No. 1020130013269 discloses a culture composition which is improved in sensitivity to a campylobacter and a preparation method thereof,

본 발명은 상기의 필요성에 의하여 안출된 것으로서, 본 발명의 목적은 개선된 캠필로박터 검출 방법을 제공하는 것이다.SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and an object of the present invention is to provide an improved method for detecting camphor lamps.

상기의 목적을 달성하기 위하여 본 발명은 실린지 필터법을 이용한 시료 내 개선된 캠필로박터 검출방법을 제공한다.In order to achieve the above object, the present invention provides an improved method for detecting a camphor bulb in a sample using a syringe filter method.

또 본 발명은 증균 전에 실린지 필터법을 실시하는 실린지 필터법을 이용한 시료 내 개선된 캠필로박터 검출방법을 제공한다.Further, the present invention provides an improved method for detecting a camphor bulb in a sample using a syringe filter method in which a syringe filter method is performed before enrichment.

본 발명의 일 구현예에 있어서, 상기 시료는 계육인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the sample is preferably chicken meat, but is not limited thereto.

또한 본 발명은 실린지 필터법을 이용한 시료 내 캠필로박터의 선택성 개선방법을 제공한다.The present invention also provides a method for improving the selectivity of a camphyllumer in a sample using a syringe filter method.

또한 본 발명은 계육을 펩톤식염완충액(Buffered peptone water)과 섞은 후, 그 린스액을 실린지 필터를 활용하여 여과한 후, 그 여과된 린스액에 볼톤 브로쓰를 섞은 후 증균배양하는 단계를 포함하는 캠필로박터의 선택성 개선방법을 제공한다.In addition, the present invention includes a step of mixing chicken meat with a peptone peptone water, filtering the rinse solution using a syringe filter, adding a bolt broth to the filtered rinse solution, To provide a method for improving the selectivity of the camphor bumper.

이하 본 발명을 설명한다.Hereinafter, the present invention will be described.

본 발명에서는 실린지 필터(syringe filtration)를 이용한 캠필로박터의 선택적 검출법을 개발하였다. 실린지 필터는 주사기(syringe)에 끼운 후, 위에서 누르는 압력을 이용하여 균을 필터사이로 통과시키는 방법이다. 실린지 필터를 이용할 경우, 시간이 매우 단축되고 오염의 가능성도 적으면서 유사한 효과를 보일 수 있다. 본 발명에서는 실린지 필터를 활용하여 캠필로박터의 선택적인 검출을 수행해 보았다.In the present invention, a selective detection method of a camphyllobum using syringe filtration has been developed. The syringe filter is a method of inserting a syringe into the syringe and passing the bacteria through the filter using the pressure from above. When using a syringe filter, the time can be greatly shortened and the possibility of contamination can be reduced with a similar effect. In the present invention, the selective detection of the camphyllobum was performed using a syringe filter.

특히 본 발명에서는 증균 전에 실린지 필터를 사용하였는데, 이렇게 배양하는 경우, 증균전에 이미 정상세균총이 억제되는 효과가 있어, 목적하는 캠필로박터 세균만 선택적으로 증균배양이 가능하다. 증균이 끝난 후 필터를 하는 경우에는 늘어난 경쟁균수로 인하여 필터시에 걸리는 물질이 많다. 그러나 증균 전에 필터를 하게 되면 균수가 적은 상태에서 필터를 하기 때문에 필터링이 쉽고 증균배양부터 캠필로박터만 선택적으로 배양되는 효과를 가질 수 있다. 본 발명에서는 증균전에 실린지 필터를 사용하고 기존 사용되는 Bolton broth에 비해 적은 양의 Bolton broth를 사용하여 기존 방법과 동등한 효과를 보일 수 있는지 테스트 해 보았다. Particularly, in the present invention, a syringe filter is used before the enrichment, and when cultured in this manner, the normal bacterial flora is already inhibited by hyperpolarisation, so that only the desired Campylobacter bacteria can be selectively enriched. When the filter is applied after the enrichment, there is a large amount of material to be filtered due to the increased number of competing bacteria. However, if the filter is used before the microbial growth, the microbial cells can be easily filtered because the microbial cells are filtered with a small number of bacteria. In the present invention, the use of a syringe filter for hyperbaric sterilization and the use of a small amount of Bolton broth compared to existing bolton broths have been tested to see if they have the same effect as the conventional method.

본 발명을 통해서 알 수 있는 바와 같이, 증균 전 실린지 필터를 이용한 개선된 캠필로박터 검출법은 기존 방법에 비해 민감도는 유사하고 선택성은 훨씬 높은 것으로 나타났다. 또한 개선된 방법은 걸리는 배양시간은 동일하면서도 사용하는 Bolton broth의 양이 훨씬 적기 때문에 더 경제적인 것으로 판단된다.
As can be seen from the present invention, the improved method of detection of camphor bugler using the pre-microbial syringe filter has a similar sensitivity and a much higher selectivity than the conventional method. The improved method is also considered to be more economical because the amount of Bolton broth used is the same but the incubation time is the same.

도 1은 본 발명의 flow chart를 요약한 도이다.
도 2는 실린지 필터의 사용과 비사용시 캠필로박터 검출능력 및 경쟁균 배재능력 비교 표이다.BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart summarizing the present invention. FIG.
FIG. 2 is a table showing the comparison between the use of the syringe filter and the ability to detect the camphor bumper and the competitor dispensing ability when the syringe filter is not used.

이하 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.
The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

실시예: 실제 계육샘플에서의 실린지 필터 효능 실험Example: Syringe filter efficacy experiment in real meat sample

실제 계육샘플에서 실린지 필터의 검출감도를 비교해 보았다. Campylobacter 가 제일 문제가 되는 식품은 닭, 칠면조 등의 가금육으로서, 본 실험에서는 시중에 유통되는 닭을 80마리 사서 증균액에서 실린지 필터를 이용하여 캠필로박터를 검출하였다. We compared the detection sensitivity of syringe filters in real meat samples. Campylobacter is the most important food for poultry such as chickens and turkeys. In this experiment, 80 of the chickens in the market were purchased and Campylobacter was detected by using a syringe filter in the diluted solution.

1. 필터를 활용한 새로운 증균배양법1. New enrichment culture using filter

계육(전육)을 400ml의 Buffered peptone water과 섞은 후, 1분간 shaking한다. 섞은 후, 린스액을 0.65μm의 실린지 필터(Sartorius, Goettingen, Germany)를 활용하여 걸러준 후, 나온 린스액 1ml에 9ml의 Bolton broth를 섞은 후 42℃에서 미호기적으로 48시간 증균배양한다. 실린지 필터의 경우, 10ml 단위의 주사기에 0.65μm 실린지 필터를 결합하여 사용하였다. 필터를 하지 않는 경우에는 증균액 1ml을 Bolton broth 9ml과 그냥 섞어준다. 증균 후, 한 백금이를 Campy-Cefex agar(CCA) 배지에 획선 도말하고 배지를 미호기적 환경에서 42℃에서 48시간 배양한다. 둥글고 편평한 반투명의 집락을 Campylobacter 의심집락으로 골라서 성상확인 및 colony PCR을 통하여, Campylobacter에 대한 확인동정을 실시한다. colony PCR은 유전학적으로 Campylobacter의 확인동정을 실시하는 방법으로 방법은 다음과 같다. 계대된 집락을 0.2ml의 멸균증류수에 풀고, 10분간 끓인 후 원심분리하고 상층액을 분리하여 template DNA로 사용한다. DNA는 PCR mix kit인 MaximeTM PCR PreMix (iNtRON biotechnology, Sungnam, Korea)와 섞어주고, forward/reverse primer를 첨가해 준 후, PCR을 돌리고 밴드사이즈를 통해 Campylobacter 양성임을 최종 확인한다. 반응조건, primer의 사용농도, primer의 시퀀스 등은 Denis et al. (Denis et al. Development of a m-PCR assay for simultaneous identification of Campylobacter jejuni and C. coli. Letters in Applied Microbiology 1999, 29, 406-410)에 나온 것과 동일하게 사용하였다. 본 실험의 전반적인 과정은 아래에 도식화 되어 있다. Chicken meat (whole meat) is mixed with 400 ml of Buffered peptone water and shaken for 1 minute. After mixing, the rinsing solution is sieved using a 0.65 μm syringe filter (Sartorius, Goettingen, Germany), 9 ml of Bolton broth is mixed with 1 ml of the rinsing solution, and the mixture is cultured at 42 ° C. for 48 hours. For the syringe filter, 0.65 μm syringe filter was used in combination with a syringe of 10 ml unit. If the filter is not used, mix 1 ml of the bacterium with 9 ml of Bolton broth. After enrichment, one platinum sample is plated on Campy-Cefex agar (CCA) medium and the medium is incubated at 42 ° C for 48 hours in a microphosphorous environment. Round pick a flat semi-transparent colonies with Campylobacter suspect colonies through the aqueous phase and determine colony PCR, performed to determine identified for Campylobacter. Colony PCR is a method to identify and identify Campylobacter genetically. Passaged colonies are dissolved in 0.2 ml of sterile distilled water, boiled for 10 minutes, centrifuged and the supernatant is separated and used as template DNA. DNA is mixed with a PCR mix kit, Maxime ™ PCR PreMix (iNtRON biotechnology, Sungnam, Korea), forward / reverse primer is added, PCR is run and final confirmation is confirmed by Campylobacter size. Reaction conditions, primer concentration, and primer sequences are described in Denis et al. (Denis et al., Development of a m-PCR assay for simultaneous identification of Campylobacter jejuni and C. coli . Letters in Applied Microbiology 1999, 29, 406-410). The overall process of this experiment is illustrated below.

기존 방법(USDA FSIS protocol)Existing method (USDA FSIS protocol)

닭의 린스(rinse) 방법 및 증균방법은 미국 농림부의 식품안전기관인 USDA FSIS의 프로토콜을 바탕으로 하였으며, 아래와 같다. 계육(전육)을 400ml의 Buffered peptone water과 섞은 후, 1분간 shaking한다. Shaking이 끝난 rinse액중 25ml을 Bolton broth (2X 농도) 25ml과 섞어준 후, 42℃에서 미호기적으로 48시간 배양한다. 배양이 끝난 후 증균액 한 백금이를 CCA 배지에 획선 도말한다. 배지를 미호기적 환경에서 42℃에서 48시간 배양한 후, 둥글고 편평한 반투명의 집락을 Campylobacter 의심집락으로 골라서 성상확인 및 위에서 언급한 colony PCR을 통하여, Campylobacter에 대한 확인동정을 실시한다.The method of rinse and the method of propagation of chicken were based on USDA FSIS protocol of US Food and Drug Administration. Chicken meat (whole meat) is mixed with 400 ml of Buffered peptone water and shaken for 1 minute. 25 ml of shaking rinse solution is mixed with 25 ml of Bolton broth (2X concentration) and incubated at 42 ° C for 48 hours. After the incubation, platinum beads that are hypertrophied are plated on CCA medium. After 48 hours at 42 ℃ from Miho term culture medium environment, through the confirmation of the pick round flat translucent colonies suspected as Campylobacter colonization properties and a colony PCR mentioned above, it is performed to confirm the identification of Campylobacter.

본 발명의 상기 계육에서의 검증 실험의 경우, Chon et al. (Comparison of three selective media and validation of the VIDAS Campylobacter assay for the detection of Campylobacter jejuni in ground beef and fresh-cut vegetables. J Food Prot. 2011. 74:456-60)에 제시된 방법을 이용하여 양성검출수를 비교함으로서 민감도를 비교 측정하였다. 또한 캠필로박터가 아닌 다른 경쟁균(competing flora)이 발견된 배지의 수를 비교하여 선택성을 비교하였다 (경쟁균이 적다는 것은 불필요한 균을 배제하는 능력이 뛰어난 것을 의미하므로, 경쟁균이 발견된 배지가 적을수록 선택성은 높음). 총 20개의 샘플 중 해당되는 수를 측정하고, GraphPad Instat software (GraphPad Software, Inc. San Diego, CA, USA)를 사용해 Fisher's exact test로 각 배지의 민감도와 선택성을 비교하였다. P value를 측정하여 값이 0.05보다 적으면 유의차가 있는 것으로 판단하였다.
In the case of the chicken test in the present invention, Chon et al. (Comparison of three selective media and validation of VIDAS Campylobacter assay for the detection of Campylobacter jejuni in ground beef and fresh-cut vegetables. J Food Prot. 2011. 74: 456-60) The sensitivity was compared by comparing. In addition, the selectivity was compared by comparing the number of media in which competing flora other than Campylobacter was found. (The less competitive bacterium means the superior ability to eliminate unnecessary bacteria, The lower the selectivity is). The sensitivity and selectivity of each medium were compared using Fisher's exact test using GraphPad Instat software (GraphPad Software, Inc., San Diego, Calif., USA). P value was measured and it was judged that there was a significant difference when the value was less than 0.05.

도 2에서 알 수 있는 바와 같이, 개선된 방법이 기존 방법에 비해 양성검출율 면에 있어서는 유의차가 나지 않는 거의 동등한 검출력을 보였다. 그러나 선택성의 측면에서는 개선된 프로토콜은 경쟁집락이 전혀 자라지 않은 반면 기존 프로토클에서는 75%의 배지에서 경쟁집락이 자라 개선된 프로토콜 훨씬 높은 선택성을 보여주는 것을 확인하였다.
As can be seen from Fig. 2, the improved method showed almost the same detection power with no significant difference in the positive detection ratio compared with the conventional method. However, in terms of selectivity, we found that the improved protocol did not grow at all, while the existing protocol showed a much higher selectivity of the improved protocol because of the growing competition of 75% of the badges.

Claims (8)

증균 전에 실린지 필터법을 이용하는 단계를 포함하는 시료 내 캠필로박터 검출방법. Using a syringe filter method before enrichment. 삭제delete 제1항에 있어서, 상기 시료는 계육인 것을 특징으로 하는 캠필로박터 검출방법. The method according to claim 1, wherein the sample is chicken meat. 제1항에 있어서, 상기 방법은 실린지 필터법을 사용하지 않은 방법에 비하여 캠필로박터에 대한 선택성이 향상된 방법. The method according to claim 1, wherein the method is improved in selectivity to a camphyllobum in comparison with a method that does not use a syringe filter method. 삭제delete 삭제delete 삭제delete 삭제delete
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* Cited by examiner, † Cited by third party
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Journal of Food Protection, Vol.45(14), pp.1332-1337(1982). *
Journal of Food Protection, Vol.45(14), pp.1332-1337(1982).*

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