WO2020116955A1 - Nucleic acid extraction method for direct analytic diagnosis of carbapenemase-producing genes from stool or rectal swab specimen - Google Patents

Nucleic acid extraction method for direct analytic diagnosis of carbapenemase-producing genes from stool or rectal swab specimen Download PDF

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WO2020116955A1
WO2020116955A1 PCT/KR2019/017083 KR2019017083W WO2020116955A1 WO 2020116955 A1 WO2020116955 A1 WO 2020116955A1 KR 2019017083 W KR2019017083 W KR 2019017083W WO 2020116955 A1 WO2020116955 A1 WO 2020116955A1
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nucleic acid
carbapenemase
buffer
acid extraction
extraction method
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박연준
이지은
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가톨릭대학교 산학협력단
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

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  • the present invention is to extract nucleic acids for the detection of carbapenemase-generating genes directly from fecal or rectal swab specimens. More specifically, the detection method is improved so that nucleic acid extraction ability is excellent and economical in terms of time and cost.
  • the present invention relates to a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a fecal or rectal swab sample.
  • Carbapenemase inhibition test Carbapenemase Nordmann-poirel test (PCR), and polymerase chain reaction (PCR) are used to detect whether carbapenemase is currently produced, but all of them must use a fungal colony. However, incubation for 18-24 hours is required until the colony is formed.
  • Xpert equipment which automatically generates PCR by simply mixing it with a sample and a lysis buffer, has been developed and used worldwide.
  • Xpert equipment which automatically generates PCR by simply mixing it with a sample and a lysis buffer
  • the present invention has been devised to solve the above problems, and the object of the present invention is to quickly and accurately stool in an easy way without using expensive equipment in a method for detecting intestinal bacteria producing carbapenemase or It is intended to provide a nucleic acid extraction method for the detection of a gene that enables the detection of carbapenemase production directly from a rectal swab sample.
  • a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a stool or rectal swab sample according to the present invention for achieving the above object is a sample for preparing a mixture formed by mixing a stool or rectal swab sample with a buffer for pretreatment Pretreatment step; A centrifugation step of centrifuging the mixture to form a supernatant; The supernatant was placed in a reagent tube containing a cell lysis buffer along with a non-specific protein removal solvent and left to form a drop mixture, and the drop mixture was placed in a reagent tube with a filter card to filter the filter card.
  • CPE carbapenemase intestinal bacteria
  • the non-specific protein removal solvent is a protease.
  • the drop mixture is formed by placing the supernatant together with the protease in a reagent tube containing a cytolytic buffer and standing at a constant temperature for a period of time.
  • the cell lysis buffer is preferably an AL buffer.
  • the filter card is an FTA card.
  • the buffer for removing impurities is an FTA reagent buffer.
  • the elution buffer is a TE buffer.
  • the nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a stool or rectal swab sample according to the present invention having the above-described configuration has superior extraction ability compared to a nucleic acid extraction method using the conventional standard method, as well as time It is very economical in terms of light cost, and it is possible to expect the advantage of improving the efficiency of nucleic acid extraction by reducing manpower or cost of using expensive equipment.
  • FIG. 1 is a flowchart of a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly in a fecal or rectal swab sample according to an embodiment of the present invention.
  • Figure 2 is a view showing the arrangement state in the reagent tube of the FTA card employed for the implementation of an embodiment of the present invention.
  • FIG. 3 is a view for explaining the drip step employed in an embodiment of the present invention.
  • FIG. 4 is a view for explaining the nucleic acid extraction step employed in an embodiment of the present invention.
  • 5 is a view for explaining the comparison of the results according to the nucleic acid extraction method of the standard method and an embodiment of the present invention.
  • FIG. 6 is a view for explaining the comparison of the Ct value according to the nucleic acid extraction method of the standard method and an embodiment of the present invention.
  • FIG. 1 is a flow chart of a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly in a fecal or rectal swab sample according to an embodiment of the present invention
  • FIG. 2 is an FTA card employed for the implementation of an embodiment of the present invention Is a diagram showing the arrangement state in the reagent tube of
  • Figure 3 is a view for explaining the dripping step employed in one embodiment of the present invention
  • Figure 4 is for explaining the nucleic acid extraction step employed in one embodiment of the present invention
  • 5 is a diagram for comparatively explaining the results of the standard method and the nucleic acid extraction method of an embodiment of the present invention
  • FIG. 6 is a diagram for comparatively explaining the standard method and the Ct value according to the nucleic acid extraction method of an embodiment of the present invention to be.
  • a nucleic acid extraction method for analytical diagnosis of a carbapenemase-producing gene directly from a fecal or rectal swab sample for example, enteric bacteria (CPE; Carbapenemase-Producing Enterobacteriacea)
  • CPE enteric bacteria
  • S1 sample pre-treatment step
  • S2 centrifugation step
  • S3 dropping step
  • S4 impurity removal step
  • S5 nucleic acid extraction step
  • sample pre-treatment step (S1) a sample is first prepared, and a process of mixing the prepared sample with a reagent is performed.
  • the sample is made of a certain amount of sample including Rectal swab or Stool, and in the sample pre-treatment step (S1), the pre-treatment mixture formed by mixing the sample with a solution such as a buffer for pre-treatment at 95° C. 5 The process of heating for a minute is performed.
  • a solution such as a buffer for pre-treatment at 95° C. 5
  • various solutions may be used as the pretreatment buffer, but in this embodiment, an ATL buffer (manufacturer; Qiagen, Hilden, Germany, model No. Cat No./ID:19076) was employed.
  • the centrifugation step (S2) performed after the sample pre-treatment step (S1) is for precipitating impurities in the mixture and obtaining a supernatant, for example, centrifugation at 12,000 rpm for 5 minutes to enable formation of a supernatant. .
  • the supernatant thus formed is placed in a reagent tube 3 containing a buffer for cell lysis together with Protease K (15ul) and left at 70° C. for 10 minutes to form a drop mixture.
  • the protease is intended to remove non-specific proteins and the like contained in the sample, and can increase the precision and efficiency of carbapenemase-generating gene detection.
  • cytolytic buffer A variety of solutions that enable cell lysis can be employed as the cytolytic buffer, but in this example, an AL buffer (manufacturer; Qiagen, Hilden, German, Model No. Cat No./ID:19075) was employed.
  • AL buffer manufactured by Qiagen, Hilden, German, Model No. Cat No./ID:19075
  • the reagent tube (3) refers to a tube made of a tubular shape to accommodate a liquid mixture, and of course, various tubes can be used, but in this embodiment, an e-tube (eppendorf tube) is employed. Became.
  • a filter card 2 made of a thin membrane structure is inserted into the reagent tube 3, and a process of dripping the drop mixture is performed as shown in FIG.
  • the filter card 2 is for capturing the nucleic acid contained in the dropping mixture, and various filters may be used, but in this embodiment, an FTA card is employed.
  • the filter card (2; FTA card) is cut into a diameter suitable for the inner diameter corresponding to the installation position of the reagent tube (3; e-tube) by a punch, and then inserted into the e-tube in a form similar to that of a manure net.
  • the FTA card 2 is pretreated paper using chemicals such as Tris, EDTA, Uric acid, and SDS dried on cellulose fiber, so that cell components can be dissolved and DNA/RNA can be preserved in the celluose fiber matrix. do.
  • the impurity removal step a process for removing impurities other than the nucleic acid to be extracted is performed after adding a buffer for removing impurities to the e-tube.
  • buffer for removing impurities various buffers capable of removing impurities other than the nucleic acid to be extracted may be employed, as well as the FTA card 2 as long as the FTA card 2 is used in the dropping step. It is preferable that FTA purification reagent buffer (GE Healthcare Life Science, Pittsburgh, USA) is employed to facilitate removal of residual impurities.
  • FTA purification reagent buffer GE Healthcare Life Science, Pittsburgh, USA
  • the impurities are removed and the FTA card 2 with the nucleic acid to be extracted remains in the other reagent tube 3, and then extracting the nucleic acid for extracting the nucleic acid Step is performed.
  • an elution buffer for eluting the extraction target is placed in the e-tube, and after a certain period of time, the FTA card 2 is removed, and then a solution in which the nucleic acid is eluted is obtained. The process is performed.
  • the solution is used for testing, nucleic acids for analytical diagnosis of carbapenemase-producing intestinal bacteria (CPE) can be smoothly extracted.
  • CPE carbapenemase-producing intestinal bacteria
  • the buffer for elution various buffers capable of elution of the nucleic acid to be extracted may be employed, and it is preferable that the buffer is a TE buffer (10 mM Tris-HCL pH 8.0; 1 mM EDTA pH 8.0) having excellent elution efficiency. .
  • the method of extracting the nucleic acid through these steps makes it possible to detect the nucleic acid to be extracted more economically and accurately and quickly than the well-known standard method (Qiagen reagent kit).
  • the standard method has the advantage of extracting clean and high concentration of nucleic acid, but the nucleic acid extraction method has many steps in 19 steps, and it takes about 30 minutes only for the pre-treatment step and about 90 minutes if it includes the washing step.
  • the sample extraction cost is about 300,000 won per 50T, that is, 6000 won per sample, which is very high considering that the PCR test reagent is 120,000 won.
  • an embodiment of the present invention is configured to extract nucleic acids using the FTA card 2, thereby deriving the advantage of extracting nucleic acids precisely and quickly at a relatively low cost.
  • the concentration of bacteria producing Carbapenemase obtained from the obtained solution was diluted stepwise from 10 ⁇ 7 CFU/ml to 10 ⁇ 1 CFU/ml, and repeated experiments at a specific concentration three times. It was judged to be detected when positive was found more than once, and the average of the Ct values was obtained and compared.
  • nucleic acids were extracted using the Qiagen kit method (standard method) and the FTA card (2) method (this example) as shown in FIG. 5 and analyzed by real-time PCR.
  • Bio-Rad SsoAdvanced Universal SYBR Green supermix kit was used as a reagent for real-time PCR analysis, and Bio-Rad CFX connect Real-time system was used as a device. 2 ul of the extracted nucleic acid was used. (6 x 10 ⁇ 5 CFU/reaction)
  • the Ct value that can compare the Qiagen kit and the FTA method and the Xpert value were additionally compared.
  • analysis was performed up to 10 ⁇ 2 CFU/rxn, and the average of Ct values was 33.91.
  • the average of Ct values at 10 ⁇ 2 CFU/rxn concentration is 32.76. It was possible to detect up to 10 ⁇ 1 CFU/rxn and the average of Ct values was 34.84.
  • Xpert's Ct values were 29.9 and 34.7, respectively.
  • a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from feces or rectal swab specimens is more capable of extracting than a conventional nucleic acid extraction method
  • the Qiagen kit used for the comparative experiment of this example is a commercial DNA/RNA extraction kit that is recognized as a reference method, and generally lysis buffer is added to vortex to dissolve cell components, and then the column containing the membrane is opened. This kit is used to extract DNA/RNA by washing.

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Abstract

The present invention is for extracting a nucleic acid for directly detecting carbapenemase-producing genes from a stool or rectal swab specimen. The present invention has excellent extractability compared to a nucleic acid extraction method by means of an existing standard scheme, has great economical advantage in terms of time and cost, and allows reduced manpower or cost from using an expensive instrument, and thus the nucleic acid extraction efficiency can be enhanced.

Description

[규칙 제26조에 의한 보정 31.12.2019]　대변 또는 직장 면봉 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법[Correction 31.12.2019 under Article 26] 핵산 Nucleic acid extraction method for analytical diagnosis of carbapenemase-generating gene directly from stool or rectal swab samples
본 발명은 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 검출을 위해 핵산을 추출하기 위한 것으로, 더욱 상세하게는 핵산 추출 능력이 우수하고 시간 및 비용 면에서 경제적일 수 있도록, 검출 방법이 개선된 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법에 관한 것이다. The present invention is to extract nucleic acids for the detection of carbapenemase-generating genes directly from fecal or rectal swab specimens. More specifically, the detection method is improved so that nucleic acid extraction ability is excellent and economical in terms of time and cost. The present invention relates to a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a fecal or rectal swab sample.
현재 카바페넴아제 생성여부를 검출하기 위해서는 카바페넴아제 억제시험, Carba NP test (Carbapenemase Nordmann-poirel test), PCR (polymerase chain reaction) 등을 이용하고 있으나, 이들은 모두 균 집락을 이용해야 한다. 그런데, 균 집락이 형성되기 까지는 18-24시간의 배양을 필요로 한다. Carbapenemase inhibition test, Carbapenemase Nordmann-poirel test (PCR), and polymerase chain reaction (PCR) are used to detect whether carbapenemase is currently produced, but all of them must use a fungal colony. However, incubation for 18-24 hours is required until the colony is formed.
carbapenemase생성 세균을 보유하는 환자의 신속한 검출을 위해서는 검체에서 Qiagen등의 kit를 이용하여 DNA를 추출하는 것과 같이 검체에서 직접 carbapenemase의 유무를 검출하는 것이 필요한데, 이러한 검출방법 방법의 경우 시간과 노력, 비용이 많이 드는 단점이 있다. For the rapid detection of patients with carbapenemase-producing bacteria, it is necessary to detect the presence or absence of carbapenemase directly in the sample, such as extracting DNA using a kit such as Qiagen from the sample. There are many disadvantages of this.
최근 이러한 단점을 극복하기 위해 검체와 lysis buffer와 혼합해 주기만 하면 PCR이 자동으로 되는 Xpert 장비(Cepheid)가 개발되어 전세계적으로 많이 이용되고 있다. 그러나, Xpert를 이용하여 검출하는 방법의 경우에도 고가의 시약 및 장비 사용으로 인하여 많은 비용이 드는 문제점이 있다. In order to overcome these shortcomings, Xpert equipment (Cepheid), which automatically generates PCR by simply mixing it with a sample and a lysis buffer, has been developed and used worldwide. However, even in the case of detection using Xpert, there is a problem that it is expensive due to the use of expensive reagents and equipment.
따라서, 본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로, 본 발명의 목적은 카바페넴아제 생성 장내세균을 검출하는 방법에 있어서 고가의 장비를 사용하지 않고도 쉬운 방법으로 신속하고 정밀하게 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성여부를 검출할 수 있게 하는 유전자의 검출을 위한 핵산 추출 방법을 제공하고자 하는 것이다. Therefore, the present invention has been devised to solve the above problems, and the object of the present invention is to quickly and accurately stool in an easy way without using expensive equipment in a method for detecting intestinal bacteria producing carbapenemase or It is intended to provide a nucleic acid extraction method for the detection of a gene that enables the detection of carbapenemase production directly from a rectal swab sample.
상기 목적을 달성하기 위한 본 발명에 의한 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법은, 대변 또는 rectal swab 검체를 전처리용 버퍼와 혼합하여 형성된 혼합물을 준비하는 검체 전처리 단계; 상기 혼합물을 원심분리하여 상층액을 형성하는 원심분리단계; 상기 상층액을 비특이 단백질 제거용제와 함께 세포용해용 버퍼가 수용되어 있는 시약용 튜브에 넣고 방치하여 점적용 혼합물을 형성시키고, 상기 점적용 혼합물을 필터카드가 있는 시약용 튜브에 넣어 그 필터카드에 점적시키는 점적단계; 상기 시약용 튜브에 불순물 제거용 버퍼를 넣은 후 추출대상인 카바페넴아제 생성장내세균(CPE)의 핵산을 제외한 나머지 불순물들을 제거하는 불순물제거단계; 및 상기 필터카드를 상기 추출대상 핵산을 용출시키기 위한 용출용 버퍼가 들어 있는 튜브에 넣고, 일정시간 경과 후 상기 필터카드를 제거한 후 상기 장내세균(CPE)의 핵산이 응집되어 있는 용액을 취득하는 핵산 추출단계;를 포함하여 이루어지는 것을 특징으로 한다. A nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a stool or rectal swab sample according to the present invention for achieving the above object is a sample for preparing a mixture formed by mixing a stool or rectal swab sample with a buffer for pretreatment Pretreatment step; A centrifugation step of centrifuging the mixture to form a supernatant; The supernatant was placed in a reagent tube containing a cell lysis buffer along with a non-specific protein removal solvent and left to form a drop mixture, and the drop mixture was placed in a reagent tube with a filter card to filter the filter card. Dropping step to drop on; An impurity removal step of adding a buffer for removing impurities to the reagent tube, and then removing the remaining impurities except for nucleic acids of carbapenemase intestinal bacteria (CPE) to be extracted; And a nucleic acid for inserting the filter card into a tube containing an elution buffer for eluting the nucleic acid to be extracted, removing the filter card after a certain period of time, and obtaining a solution in which nucleic acids of the intestinal bacteria (CPE) are aggregated. It characterized in that it comprises a; extraction step.
상기 비특이 단백질 제거용제는 프로테아제인 것이 바람직하다. It is preferable that the non-specific protein removal solvent is a protease.
상기 점적용 혼합물은, 상기 상층액을 상기 프로테아제와 함께 세포용해성 버퍼가 수용되어 있는 시약용 튜브에 넣고 일정 온도에서 일정 시간 동안 방치함으로써 형성되는 것이 바람직하다. Preferably, the drop mixture is formed by placing the supernatant together with the protease in a reagent tube containing a cytolytic buffer and standing at a constant temperature for a period of time.
상기 세포용해용 버퍼는, AL buffer인 것이 바람직하다. The cell lysis buffer is preferably an AL buffer.
상기 필터카드는 FTA 카드인 것이 바람직하다.Preferably, the filter card is an FTA card.
상기 불순물 제거용 버퍼는 FTA reagent buffer인 것이 바람직하다. It is preferable that the buffer for removing impurities is an FTA reagent buffer.
상기 용출용 버퍼는 TE buffer인 것이 바람직하다. It is preferable that the elution buffer is a TE buffer.
상술한 바와 같은 구성을 가지는 본 발명에 의한 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법은, 기존의 표준법에 의한 핵산 추출방법보다 추출 능력이 우수함은 물론, 시간 빛 비용면에서 매우 경제적인 장점과, 고가의 장비 사용에 따른 인력이나 비용을 줄일 수 있어서 핵산 추출의 효율성을 향상시킬 수 있는 장점을 기대할 수 있게 한다. The nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a stool or rectal swab sample according to the present invention having the above-described configuration has superior extraction ability compared to a nucleic acid extraction method using the conventional standard method, as well as time It is very economical in terms of light cost, and it is possible to expect the advantage of improving the efficiency of nucleic acid extraction by reducing manpower or cost of using expensive equipment.
도 1은 본 발명의 일실시예에 따른 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법의 흐름도.1 is a flowchart of a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly in a fecal or rectal swab sample according to an embodiment of the present invention.
도 2는 본 발명 일실시예의 구현을 위해 채용된 FTA 카드의 시약용 튜브 내 배치상태를 보인 도면.Figure 2 is a view showing the arrangement state in the reagent tube of the FTA card employed for the implementation of an embodiment of the present invention.
도 3은 본 발명 일실시예에 채용된 점적단계를 설명하기 위한 도면.3 is a view for explaining the drip step employed in an embodiment of the present invention.
도 4는 본 발명 일실시예에 채용된 핵산 추출단계를 설명하기 위한 도면.4 is a view for explaining the nucleic acid extraction step employed in an embodiment of the present invention.
도 5는 표준법과 본 발명 일실시예의 핵산추출방법에 따른 결과를 비교 설명하기 위한 도면.5 is a view for explaining the comparison of the results according to the nucleic acid extraction method of the standard method and an embodiment of the present invention.
도 6은 표준법과 본 발명 일실시예의 핵산추출방법에 따른 Ct값을 비교 설명하기 위한 도면.6 is a view for explaining the comparison of the Ct value according to the nucleic acid extraction method of the standard method and an embodiment of the present invention.
이하의 설명에서 본 발명에 대한 이해를 명확히 하기 위하여, 본 발명의 특징에 대한 공지의 기술에 대한 설명은 생략하기로 한다. 이하의 실시 예는 본 발명의 이해를 돕기 위한 상세한 설명이며, 본 발명의 권리 범위를 제한하는 것이 아님은 당연할 것이다. 따라서, 본 발명과 동일한 기능을 수행하는 균등한 발명 역시 본 발명의 권리 범위에 속할 것이다.In the following description, in order to clarify the understanding of the present invention, descriptions of well-known technologies for the features of the present invention will be omitted. The following examples are detailed descriptions to help the understanding of the present invention, and it will be understood that the scope of the present invention is not limited. Accordingly, an equivalent invention that performs the same function as the present invention will also fall within the scope of the present invention.
그리고, 이하의 설명에서 동일한 식별 기호는 동일한 구성을 의미하며, 불필요한 중복적인 설명 및 공지 기술에 대한 설명은 생략하기로 한다. 또한, 상기 발명의 배경이 되는 기술에 대한 기재 내용과 중복되는 이하의 본 발명의 각 실시예에 관한 설명 역시 생략하기로 한다.And, in the following description, the same identification symbol means the same configuration, and unnecessary redundant description and description of known technology will be omitted. In addition, the description of each embodiment of the present invention that overlaps with the description of the technology that is the background of the present invention will also be omitted.
이하에서는 본 발명의 일실시예에 따른 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법을 첨부된 도면을 참조하여 상세히 설명하기로 한다 .Hereinafter, a nucleic acid extraction method for analytical diagnosis of a carbapenemase generating gene directly from a feces or rectal swab sample according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.
도 1은 본 발명의 일실시예에 따른 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법의 흐름도이고, 도 2는 본 발명 일실시예의 구현을 위해 채용된 FTA 카드의 시약용 튜브 내 배치상태를 보인 도면이며, 도 3은 본 발명 일실시예에 채용된 점적단계를 설명하기 위한 도면이며, 도 4는 본 발명 일실시예에 채용된 핵산 추출단계를 설명하기 위한 도면이며, 도 5는 표준법과 본 발명 일실시예의 핵산추출방법에 따른 결과를 비교 설명하기 위한 도면이며, 도 6은 표준법과 본 발명 일실시예의 핵산추출방법에 따른 Ct값을 비교 설명하기 위한 도면이다. 1 is a flow chart of a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly in a fecal or rectal swab sample according to an embodiment of the present invention, and FIG. 2 is an FTA card employed for the implementation of an embodiment of the present invention Is a diagram showing the arrangement state in the reagent tube of, Figure 3 is a view for explaining the dripping step employed in one embodiment of the present invention, Figure 4 is for explaining the nucleic acid extraction step employed in one embodiment of the present invention 5 is a diagram for comparatively explaining the results of the standard method and the nucleic acid extraction method of an embodiment of the present invention, and FIG. 6 is a diagram for comparatively explaining the standard method and the Ct value according to the nucleic acid extraction method of an embodiment of the present invention to be.
도 1에 잘 도시된 바와 같이, 본 발명의 일실시예에 따른 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법은, 예컨대 장내세균(CPE;Carbapenemase-Producing Enterobacteriacea)과 같은 유전자에서 카바페넴아제 생성여부를 검출하기 위한 것으로, 검체 전처리 단계(S1)와 원심분리단계(S2)와 점적단계(S3)와 불순물제거단계(S4)와 핵산 추출단계(S5)를 포함하여 이루어진다.1, a nucleic acid extraction method for analytical diagnosis of a carbapenemase-producing gene directly from a fecal or rectal swab sample according to an embodiment of the present invention, for example, enteric bacteria (CPE; Carbapenemase-Producing Enterobacteriacea) To detect whether carbapenemase is generated from the same gene, and includes a sample pre-treatment step (S1), a centrifugation step (S2), a dropping step (S3), an impurity removal step (S4), and a nucleic acid extraction step (S5). Is done by
상기 검체 전처리 단계(S1)에서는 먼저 샘플을 준비하고, 그 준비된 샘플을 시약과 혼합하는 과정이 수행된다. In the sample pre-treatment step (S1), a sample is first prepared, and a process of mixing the prepared sample with a reagent is performed.
즉, 상기 샘플은 Rectal swab 또는 Stool을 포함한 일정량의 검체로 이루어지고, 상기 검체 전처리 단계(S1)에서는 상기 검체를 전처리용 버퍼(buffer)와 같은 용액과 혼합하여 형성된 전처리용 혼합물을 95℃에서 5분간 가열하는 과정이 수행된다. 여기서, 상기 전처리용 버퍼는 다양한 용액이 사용될 수 있으나, 본 실시예에서는 ATL 버퍼(제조사;Qiagen, Hilden,Germany, 모델명 Cat No./ID:19076)가 채용되었다. That is, the sample is made of a certain amount of sample including Rectal swab or Stool, and in the sample pre-treatment step (S1), the pre-treatment mixture formed by mixing the sample with a solution such as a buffer for pre-treatment at 95° C. 5 The process of heating for a minute is performed. Here, various solutions may be used as the pretreatment buffer, but in this embodiment, an ATL buffer (manufacturer; Qiagen, Hilden, Germany, model No. Cat No./ID:19076) was employed.
이러한 검체 전처리 단계(S1) 이후 수행되는 원심분리단계(S2)는, 상기 혼합물에서 불순물을 침전시키고 상층액을 얻기 위한 것으로, 예컨대 12,000 rpm에서 5분동안 원심분리하여 상층액의 형성을 가능하게 한다. The centrifugation step (S2) performed after the sample pre-treatment step (S1) is for precipitating impurities in the mixture and obtaining a supernatant, for example, centrifugation at 12,000 rpm for 5 minutes to enable formation of a supernatant. .
이와 같이 형성된 상층액을 프로테아제(Protease K;15ul)와 함께 세포용해용 버퍼(buffer)가 수용되어 있는 시약용 튜브(3)에 넣고 70℃ 에서 10분 동안 방치하여 점적용 혼합물을 형성시킨다. The supernatant thus formed is placed in a reagent tube 3 containing a buffer for cell lysis together with Protease K (15ul) and left at 70° C. for 10 minutes to form a drop mixture.
상기 프로테아제는 상기 검체에 포함된 비특이단백질 등을 제거하기 위한 것으로, 카바페넴아제 생성 유전자 검출의 정밀성과 효율성을 높일 수 있게 한다. The protease is intended to remove non-specific proteins and the like contained in the sample, and can increase the precision and efficiency of carbapenemase-generating gene detection.
상기 세포용해성 버퍼로는 세포 용해를 가능하게 하는 다양한 용액이 채용될 수 있으나, 본 실시예에서는 AL buffer(제조사;Qiagen, Hilden,Germany, 모델명 Cat No./ID:19075)가 채용되었다. A variety of solutions that enable cell lysis can be employed as the cytolytic buffer, but in this example, an AL buffer (manufacturer; Qiagen, Hilden, German, Model No. Cat No./ID:19075) was employed.
또한, 상기 시약용 튜브(3)는 액체 혼합물을 수용할 수 있도록 통 형상으로 이루어진 튜브를 의미하는 것으로, 다양한 튜브가 사용될 수 있음은 물론이나, 본 실시예에서는 e-tube(eppendorf tube)가 채용되었다. In addition, the reagent tube (3) refers to a tube made of a tubular shape to accommodate a liquid mixture, and of course, various tubes can be used, but in this embodiment, an e-tube (eppendorf tube) is employed. Became.
상기 점적단계에서는, 도 2와 같이 상기 시약용 튜브(3)에 얇은 막 구조로 이루어진 필터카드(2)를 넣고, 도 3과 같이 상기 점적용 혼합물을 점적하는 과정이 수행된다. In the dropping step, as shown in FIG. 2, a filter card 2 made of a thin membrane structure is inserted into the reagent tube 3, and a process of dripping the drop mixture is performed as shown in FIG.
상기 필터카드(2)는, 상기 점적용 혼합물에 함유된 핵산을 포집하기 위한 것으로, 다양한 필터가 사용될 수 있으나, 본 실시예에서는 FTA 카드가 채용되었다. The filter card 2 is for capturing the nucleic acid contained in the dropping mixture, and various filters may be used, but in this embodiment, an FTA card is employed.
상기 필터카드(2;FTA 카드)는 펀치에 의해 상기 시약용 튜브(3;e-tube)의 설치위치에 해당하는 내경에 적합한 직경으로 잘라진 후 대략 거름망과 유사한 형태로 e-tube에 삽입된다. The filter card (2; FTA card) is cut into a diameter suitable for the inner diameter corresponding to the installation position of the reagent tube (3; e-tube) by a punch, and then inserted into the e-tube in a form similar to that of a manure net.
여기서, 상기 FTA 카드(2)는 Cellulose fiber에 건조된 Tris, EDTA, Uric acid, SDS 등 화학물질을 이용한 전처리한 종이로, 세포 성분을 용해시키고, DNA/RNA를 celluose fiber matrix내에 보존할 수 있게 한다. Here, the FTA card 2 is pretreated paper using chemicals such as Tris, EDTA, Uric acid, and SDS dried on cellulose fiber, so that cell components can be dissolved and DNA/RNA can be preserved in the celluose fiber matrix. do.
한편, 상기 불순물제거단계에서는, 상기 e-tube에 불순물 제거용 버퍼를 넣은 후 추출대상인 핵산을 제외한 나머지 불순물들을 제거하는 과정이 수행된다. On the other hand, in the impurity removal step, a process for removing impurities other than the nucleic acid to be extracted is performed after adding a buffer for removing impurities to the e-tube.
상기 불순물 제거용 버퍼로는 추출대상 핵산을 제외한 나머지 불순물을 제거할 수 있는 다양한 buffer가 채용될 수 있음은 물론이나, 상기 점적단계에서 FTA 카드(2)가 사용된 이상 그 FTA 카드(2)에 잔류한 불순물 제거가 용이하도록 FTA purification reagent buffer(GE Healthcare Life Science,Pittsburgh,USA)가 채용되는 것이 바람직하다. As the buffer for removing impurities, various buffers capable of removing impurities other than the nucleic acid to be extracted may be employed, as well as the FTA card 2 as long as the FTA card 2 is used in the dropping step. It is preferable that FTA purification reagent buffer (GE Healthcare Life Science, Pittsburgh, USA) is employed to facilitate removal of residual impurities.
이와 같이, 상기 점적단계와 불순물 제거단계를 거친 후, 불순물이 제거되고 추출대상 핵산이 남아 있게 된 FTA 카드(2)를 다른 시약용 튜브(3)에 넣은 후, 상기 핵산 을 추출하기 위한 핵산 추출단계가 수행된다. Thus, after the dropping step and the impurity removing step, the impurities are removed and the FTA card 2 with the nucleic acid to be extracted remains in the other reagent tube 3, and then extracting the nucleic acid for extracting the nucleic acid Step is performed.
상기 핵산 추출단계에서는, 도 4와 같이 상기 추출대상을 용출시키기 위한 용출용 버퍼를 상기 e-tube에 넣고, 일정시간 경과 후 상기 FTA 카드(2)를 제거한 후 상기 핵산이 용출되어 있는 용액을 취득하는 과정이 수행된다. 본 실시예는 이 용액을 검사에 사용하게 됨에 따라 카바페넴아제 생성 장내세균(CPE)의 분석적 진단을 위한 핵산을 원활하게 추출할 수 있게 된다. In the nucleic acid extraction step, as shown in FIG. 4, an elution buffer for eluting the extraction target is placed in the e-tube, and after a certain period of time, the FTA card 2 is removed, and then a solution in which the nucleic acid is eluted is obtained. The process is performed. In this embodiment, as the solution is used for testing, nucleic acids for analytical diagnosis of carbapenemase-producing intestinal bacteria (CPE) can be smoothly extracted.
상기 용출용 버퍼로는 상기 추출대상 핵산의 용출을 가능하게 하는 다양한 buffer가 채용될 수 있음은 물론이나, 용출 효율이 우수한 TE buffer(10mM Tris-HCL pH 8.0;1mM EDTA pH 8.0) 인 것이 바람직하다. As the buffer for elution, various buffers capable of elution of the nucleic acid to be extracted may be employed, and it is preferable that the buffer is a TE buffer (10 mM Tris-HCL pH 8.0; 1 mM EDTA pH 8.0) having excellent elution efficiency. .
이러한 단계를 거쳐 핵산을 추출하는 방법은 널리 알려진 표준법(Qiagen 시약키트)보다 추출대상 핵산을 경제적이면서도 정밀하고 신속하게 검출할 수 있게 한다. The method of extracting the nucleic acid through these steps makes it possible to detect the nucleic acid to be extracted more economically and accurately and quickly than the well-known standard method (Qiagen reagent kit).
즉, 상기 표준법은 깨끗하고 높은 농도의 핵산을 추출하는 장점을 가지지만 핵산 추출방법이 19 단계로 많고 전처리 단계만 약 30분이 걸리며 세척 단계까지 포함하면 약 90분이 걸리므로 핵산 추출 시간이 오래 걸린다는 단점이 있다. 또한, 검체 추출 비용이 50T에 약 30만원, 즉 1 검체당 6000원으로서 PCR 검사 시약이 1-2 만원임을 감안하면 매우 높은 편이다. That is, the standard method has the advantage of extracting clean and high concentration of nucleic acid, but the nucleic acid extraction method has many steps in 19 steps, and it takes about 30 minutes only for the pre-treatment step and about 90 minutes if it includes the washing step. There are disadvantages. In addition, the sample extraction cost is about 300,000 won per 50T, that is, 6000 won per sample, which is very high considering that the PCR test reagent is 120,000 won.
본 발명의 일실시예는 이러한 표준법과는 달리 FTA 카드(2)를 이용하여 핵산을 추출할 수 있도록 구성됨으로써, 상대적으로 저렴한 비용으로 정밀하고 신속하게 핵산을 추출할 수 있는 장점을 도출한다. Unlike the standard method, an embodiment of the present invention is configured to extract nucleic acids using the FTA card 2, thereby deriving the advantage of extracting nucleic acids precisely and quickly at a relatively low cost.
한편, 상기 추출단계에서 취득된 용액을 이용한 구체적인 분석방법은 아래와 같다. Meanwhile, a specific analysis method using the solution obtained in the extraction step is as follows.
즉, 상기 취득된 용액에서 얻어진 Carbapenemase를 생성하는 세균의 농도를 10^7 CFU/ml부터 10^1 CFU/ml까지 단계 희석하여 특정 농도에서 3회 반복 실험하였다. 2회이상 양성이 나온 경우 검출된 것으로 판정하였으며, Ct값의 평균을 구하여 비교하였다. That is, the concentration of bacteria producing Carbapenemase obtained from the obtained solution was diluted stepwise from 10^7 CFU/ml to 10^1 CFU/ml, and repeated experiments at a specific concentration three times. It was judged to be detected when positive was found more than once, and the average of the Ct values was obtained and compared.
본 실시예와 비교 실험을 위해 도 5와 같이 동일한 샘플을 Qiagen kit 방법(표준법)과 FTA 카드(2) 방법(본 실시예)으로 핵산을 추출하여 Real-time PCR로 분석을 하였다. Real-time PCR 분석을 위한 시약으로는 Bio-Rad SsoAdvanced Universal SYBR Green supermix kit가 사용되었고, 장비로는 Bio-Rad CFX connect Real-time system이 사용되었다. 추출한 핵산은 2ul를 사용하였다. (6 x 10^5 CFU/reaction)For comparative experiments with this example, nucleic acids were extracted using the Qiagen kit method (standard method) and the FTA card (2) method (this example) as shown in FIG. 5 and analyzed by real-time PCR. Bio-Rad SsoAdvanced Universal SYBR Green supermix kit was used as a reagent for real-time PCR analysis, and Bio-Rad CFX connect Real-time system was used as a device. 2 ul of the extracted nucleic acid was used. (6 x 10^5 CFU/reaction)
또한, 도 6과 같이, Qiagen kit와 FTA 방법을 비교할 수 있는 Ct값과 추가적으로 Xpert 값을 비교하였다. Qiagen kit를 이용한 경우 10^2 CFU/rxn까지 분석이 되었고 Ct 값의 평균은 33.91이었다. 본 실시예의 경우 10^2 CFU/rxn 농도에서의 Ct 값의 평균은 32.76. 이었으며, 10^1 CFU/rxn까지 검출 가능하였으며 Ct 값의 평균은 34.84 였다. 10^3 CFU/rxn 및 10^1 CFU/rxn 농도에서 Xpert의 Ct값은 각각 29.9, 34.7 이었다.In addition, as shown in FIG. 6, the Ct value that can compare the Qiagen kit and the FTA method and the Xpert value were additionally compared. In case of using Qiagen kit, analysis was performed up to 10^2 CFU/rxn, and the average of Ct values was 33.91. In the case of this example, the average of Ct values at 10^2 CFU/rxn concentration is 32.76. It was possible to detect up to 10^1 CFU/rxn and the average of Ct values was 34.84. At 10^3 CFU/rxn and 10^1 CFU/rxn concentrations, Xpert's Ct values were 29.9 and 34.7, respectively.
이러한 실험 데이터로 확인되는 바와 같이, 본 발명의 일실시예에 따른 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법은, 기존의 표준법에 의한 핵산 추출방법보다 추출 능력이 우수함은 물론, 시간 빛 비용면에서 매우 경제적인 장점과, 고가의 장비 사용에 따른 인력이나 비용을 줄일 수 있어서 핵산 추출의 효율성을 향상시킬 수 있는 장점을 기대할 수 있게 한다.As confirmed by these experimental data, a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from feces or rectal swab specimens according to an embodiment of the present invention is more capable of extracting than a conventional nucleic acid extraction method In addition to this excellence, it is possible to expect a very economical advantage in terms of time and light cost, and an advantage of improving the efficiency of nucleic acid extraction by reducing manpower or cost of using expensive equipment.
한편, 본 실시예의 비교 실험을 위해 사용되는 Qiagen kit는 commercial DNA/RNA extraction kit로 reference method로 인정되는 방법으로, 일반적으로 lysis buffer를 넣고 vortex 하여 세포 성분등을 용해 시킨 후 membrane이 들어있는 column을 이용하여 washing 하여 DNA/RNA를 추출하는 키트이다.On the other hand, the Qiagen kit used for the comparative experiment of this example is a commercial DNA/RNA extraction kit that is recognized as a reference method, and generally lysis buffer is added to vortex to dissolve cell components, and then the column containing the membrane is opened. This kit is used to extract DNA/RNA by washing.
이상 본 발명의 다양한 실시예에 대하여 설명하였으나, 본 실시예 및 본 명세서에 첨부된 도면은 본 발명에 포함되는 기술적 사상의 일부를 명확하게 나타내고 있는 것에 불과하며, 본 발명의 명세서 및 도면에 포함된 기술적 사상의 범위 내에서 당업자가 용이하게 유추할 수 있는 변형 예와 구체적인 실시예는 모두 본 발명의 권리범위에 포함되는 것이 자명하다고 할 것이다. The various embodiments of the present invention have been described above, but the drawings attached to the present embodiment and the present specification merely show a part of the technical spirit included in the present invention, and are included in the specification and the drawings of the present invention. It will be apparent that modifications and specific embodiments that can be easily inferred by those skilled in the art within the scope of the technical idea are included in the scope of the present invention.

Claims (7)

  1. 대변 또는 rectal swab 검체를 전처리용 버퍼와 혼합하여 형성된 혼합물을 준비하는 검체 전처리 단계; A sample pre-treatment step of preparing a mixture formed by mixing a stool or rectal swab sample with a pre-treatment buffer;
    상기 혼합물을 원심분리하여 상층액을 형성하는 원심분리단계; A centrifugation step of centrifuging the mixture to form a supernatant;
    상기 상층액을 비특이 단백질 제거용제와 함께 세포용해용 버퍼가 수용되어 있는 시약용 튜브에 넣고 방치하여 점적용 혼합물을 형성시키고, 상기 점적용 혼합물을 필터카드가 있는 시약용 튜브에 넣어 그 필터카드에 점적시키는 점적단계; The supernatant was placed in a reagent tube containing a cell lysis buffer along with a non-specific protein removal solvent and left to form a drop mixture, and the drop mixture was placed in a reagent tube with a filter card to filter the filter card. Dropping step to drop on;
    상기 시약용 튜브에 불순물 제거용 버퍼를 넣은 후 추출대상인 카바페넴아제 생성장내세균(CPE)의 핵산을 제외한 나머지 불순물들을 제거하는 불순물제거단계; 및 상기 필터카드를 상기 추출대상 핵산을 용출시키기 위한 용출용 버퍼가 들어 있는 튜브에 넣고, 일정시간 경과 후 상기 필터카드를 제거한 후 상기 장내세균(CPE)의 핵산이 응집되어 있는 용액을 취득하는 핵산 추출단계;를 포함하여 이루어지는 것을 특징으로 하는 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법.An impurity removal step of adding a buffer for removing impurities to the reagent tube, and then removing the remaining impurities except for nucleic acids of carbapenemase intestinal bacteria (CPE) to be extracted; And a nucleic acid for inserting the filter card into a tube containing an elution buffer for eluting the nucleic acid to be extracted, removing the filter card after a certain period of time, and obtaining a solution in which nucleic acids of the intestinal bacteria (CPE) are aggregated. Extraction step; nucleic acid extraction method for analytical diagnosis of carbapenemase-generating gene directly from feces or rectal swab specimen, characterized in that it comprises a.
  2. 제1항에 있어서,According to claim 1,
    상기 비특이 단백질 제거용제는 프로테아제인 것을 특징으로 하는 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법.The non-specific protein removal solvent is a protease, nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a stool or rectal swab sample.
  3. 제2항에 있어서,According to claim 2,
    상기 점적용 혼합물은, 상기 상층액을 상기 프로테아제와 함께 세포용해성 버퍼가 수용되어 있는 시약용 튜브에 넣고 일정 온도에서 일정 시간 동안 방치함으로서 형성되는 것을 특징으로 하는 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법.The drop-in mixture is formed by placing the supernatant together with the protease in a reagent tube containing a cytolytic buffer and standing for a period of time at a constant temperature. Nucleic acid extraction method for the analytical diagnosis of the gene produced.
  4. 제1항에 있어서,According to claim 1,
    상기 세포용해용 버퍼는, AL buffer인 것을 특징으로 하는 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법.The cell lysis buffer is a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a stool or rectal swab specimen, characterized in that it is an AL buffer.
  5. 제1항에 있어서,According to claim 1,
    상기 필터카드는 FTA 카드인 것을 특징으로 하는 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법.The filter card is a FTA card, nucleic acid extraction method for analytical diagnosis of carbapenemase-generating genes directly from fecal or rectal swab specimens.
  6. 제1항에 있어서,According to claim 1,
    상기 불순물 제거용 버퍼는 FTA reagent buffer인 것을 특징으로 하는 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법.The impurity removal buffer is a FTA reagent buffer, nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a fecal or rectal swab sample.
  7. 제1항에 있어서,According to claim 1,
    상기 용출용 버퍼는 TE buffer 인 것을 특징으로 하는 대변 또는 rectal swab 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법.The elution buffer is a nucleic acid extraction method for analytical diagnosis of a carbapenemase-generating gene directly from a stool or rectal swab specimen, characterized in that it is a TE buffer.
PCT/KR2019/017083 2018-12-05 2019-12-05 Nucleic acid extraction method for direct analytic diagnosis of carbapenemase-producing genes from stool or rectal swab specimen WO2020116955A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120138766A (en) * 2010-02-16 2012-12-26 사이시스 다이애그노스틱스, 인크. Nucleic acid extraction from complex matrices
KR20140000810A (en) * 2012-06-25 2014-01-06 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) Immunomagnetic capture method to rapid detect mycobacterium avium subsp. paratuberculosis in fecal sample
US20180057808A1 (en) * 2016-09-01 2018-03-01 Wisconsin Alumni Research Foundation Methods and systems for isolating and identifying nucleic acid from a plurality of microorganisms and viruses
US20180274018A1 (en) * 2017-03-27 2018-09-27 Imicroq, S.L. Rapid pcr method for detection of pathogenic microorganisms

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120138766A (en) * 2010-02-16 2012-12-26 사이시스 다이애그노스틱스, 인크. Nucleic acid extraction from complex matrices
KR20140000810A (en) * 2012-06-25 2014-01-06 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) Immunomagnetic capture method to rapid detect mycobacterium avium subsp. paratuberculosis in fecal sample
US20180057808A1 (en) * 2016-09-01 2018-03-01 Wisconsin Alumni Research Foundation Methods and systems for isolating and identifying nucleic acid from a plurality of microorganisms and viruses
US20180274018A1 (en) * 2017-03-27 2018-09-27 Imicroq, S.L. Rapid pcr method for detection of pathogenic microorganisms

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NDZI, E. S. ET AL.: "Fast Technology Analysis (FTA) Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians", JOURNAL OF EUKARYOTIC MICROBIOLOGY, vol. 63, no. 2, March 2016 (2016-03-01), pages 146 - 152, XP055717250 *

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