• 한국식품저장유통학회지
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• 제16권6호
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• pp.965-970
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• 2009

유통중인 계란의병원성균 오염상태를 시판되는 multiplex polymerase chain reaction (mPCR) kit로 검사한 결과, 총 90개의 검사시료 가운데 한 가지 이상의 미생물이 검출된 계란시료는 30개로, 검사시료의 미생물 오염도는 약 33.3% 수준이었다. 미생물별 검출 빈도는 B. cereus가 17개 시료(18.9%)에서 검출되어 가장 높은 빈도를 보였으며 Y. enterocolitica는 16개(17.8%), L. monocytogenes 15개(16.7%), St. aureus 12개(13.3%), E. coli O157:H7 4개(4.4%)의 검출빈도를 보였다. 한편, 선택배지를 이용한 viable cell count 방법에서는 27개의 시료에서 미생물이 검출되어 검사시료의 미생물 오염도는 약 30.0% 수준이였으며, 미생물별 검출 빈도는 B. cereus가 17개 시료(18.9%)에서 14개(15.6%) 시료로, Y. enterocolitica는 16개(17.8%)에서 12개(13.3%)로, L. monocytogenes는 15개(16.7%)에서 13개(14.4%)로, St. aureus는 12개(13.3%)에서 10개(11.1%)로 감소하였으며, E. coli O157:H7은 두 가지 검출방법간의 차이가 나타나지 않았다. 검출 대상 미생물 9종 가운데 Campylobacter jejuni, Vibrio parahaemolyticus, Salmonella spp., 및 Shigella spp.는 두 방법 모두에서 검출되지 않았다. 이상의 결과에서 살펴본 바와 같이 PCR을 이용한 병원성 미생물의 검출 방법은 viable cell count 방법보다 감도가 높음을 알 수 있었다.

• 한국동물위생학회지
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• 제38권1호
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• pp.37-42
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• 2015

The aim of the present study was to compare the non-invasive methods for the diagnosis of H. felis with HpSA kit-based detection method and H. felis-specific PCR assay with dog's stool samples without sacrifice. Male Beagle dogs (n=6) were infected with H. felis ATCC 49179 ($1.0{\times}10^9CFU/dog$) by intra-gastric inoculation two times at 3-day intervals, and the stool specimens of dogs were collected 1, 3, 5, 7, 14, 21 days after infection to submit to HpSA test and H. felis-specific PCR. As the results, the sensitivity of the HpSA and the PCR analysis was 50.0%, 83.3% respectively. Although HpSA test is less sensitive, it could be used for rapid, cheap and easy screening assay for H. felis infection in dog and cats. We suggest that the H. pylori stool antigen kit, HpSA, is useful and effective for monitoring H. felis infection. If HpSA test would be made with H. felis antibodies in the future, its sensitivity could be increased. Also, PCR assay could be successfully used to detect the H. felis in stools. Applying the H. pylori stool antigen kit and PCR assay may be the recommended non-invasive strategy to identify H. felis in dog and cats.

• Tuberculosis and Respiratory Diseases
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• 제49권3호
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• pp.281-289
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• 2000

배경 : 결핵의 진단에 있어 결핵균 배양검사는 결핵의 확진 검사이지만 배양에 최소한 6-8주가 소요되어 진단 및 치료가 지연되는 단점이 있다. PCR 법은 신속하고 예민하게 결핵균을 검출할 수 있지만 위양성율과 위음성율이 높아 문제시 되고 있다. 최근에 개발된 LCR법과 PCR-Hybridization은 PCR 법 보다도 민감하게 결핵균을 검출할 수 있는 것으로 알려져 있다. 이에 저자들은 하상균 배양을 기준으로 in house PCR, LCR 및 PCR- Hybridization 각각의 임상적 유용성을 알아보고자 한다. 방법 : 1998년 8월에 결핵진단을 위해 세브란스 병원 임상 병리과에 의뢰된 75검체를 대상으로 AFB 도말 염색 검사, 결핵균 배양 검사(3% Ogawa 배지, 8주간), in house PCR, LCR(Abbott LCx kit) 및 PCR Hybridization을 시행하여 각각의 민간도와 특이도를 평가해보았다. 결과 : 항산균 배양법을 기준으로 판단할 때 in house PCR, LCR(Abbott LCx kit) 및 PCR-Hybridization의 민감도는 각각 40%, 80%, 100%였고 특이도는 98.6%, 94.3%, 94.3%였다. 결론 : LCR법과 PCR-Hybridization은 결핵균 검출에 있어 우수한 민감도와 특이도를 보여 결핵의 조기진단에 유용할 것으로 사료된다.

• Journal of Veterinary Science
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• 제23권4호
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• pp.51.1-51.10
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• 2022

Background: Due to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever. Objectives: The objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV. Methods: LMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples. Results: Our results showed that the LMTIA assay could detect the 10⁴ dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%. Conclusions: The LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.

• Parasites, Hosts and Diseases
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• 제57권1호
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• pp.27-31
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• 2019

PCR is known to be the most sensitive method for diagnosing Trichomonas vaginalis infections. This study aimed to compare the sensitivity of a PCR assay for trichomoniasis (HY-PCR) developed in Hanyang University with the use of a Seeplex Ace Detection $Kit^{(R)}$, using urine collected from four Korean men with prostatic disease. Overall, HY-PCR was more sensitive than the Seeplex Kit. The use of Chelex 100 is recommended for DNA isolation in order to increase the sensitivity of the PCR test.

• Parasites, Hosts and Diseases
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• 제52권3호
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• pp.263-271
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• 2014

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. $QIAamp^{(R)}$ DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume ($50-100{\mu}l$) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ${\approx}2$ oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

• Journal of Microbiology and Biotechnology
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• 제20권10호
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• pp.1450-1456
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• 2010

Accurate and rapid diagnosis of Pandemic Influenza A/H1N1 2009 virus (H1N1 2009) infection is important for the prevention and control of influenza epidemics and the timely initiation of antiviral treatment. This study was conducted to evaluate the performance of several diagnostic tools for the detection of H1N1 2009. Flocked nasopharyngeal swabs were collected from 254 outpatients of suspected H1N1 2009 during October 2009. This study analyzed the performances of the RealTime Ready Inf A/H1N1 Detection Set (Roche), Influenza A (H1N1) Real-Time Detection Kit (Bionote), Seeplex Influenza A/B OneStep Typing Set [Seeplex Reverse Transcriptase PCR (RT-PCR)], BinaxNow Influenza A & B Test Kit [Binax Rapid Antigen Test (RAT)], and SD BIOLINE Influenza Ag kit (SD RAT). Roche and Bionote real-time RT-PCR showed identical results for the H1N1 2009 hemagglutinin gene. Compared with real-time RT-PCR, the sensitivities and specificities were 83.7% and 100% for Seeplex RT-PCR, 64.5% and 94.7% for Binax RAT, and 69.5% and 100% for SD RAT. The sensitivities of Seeplex RT-PCR, Binax RAT, and SD RAT in patients aged over 21 years were 73.7%, 47.4%, and 57.9%, respectively. The sensitivities of Seeplex RT-PCR, Binax RAT, and SD RAT on the day of initial symptoms were mostly lower (68.8%, 56.3%, and 31.3%, respectively). In conclusion, multiplex RT-PCR and RAT for the detection of H1N1 2009 were significantly less sensitive than real-time RT-PCR. Moreover, a negative RAT may require more sensitive confirmatory assays, because it cannot be ruled out from influenza infection.

• 한국동물위생학회지
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• 제37권4호
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• pp.253-261
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• 2014

Porcine circovirus disease (PCVD) is a major problem of swine industry worldwide, and diagnosis of PCV2, causal agent of PCVD, has been doing in clinical laboratories of pig disease by polymerase chain reaction (PCR) methods. But the PCR analyses have a serious problem of misdiagnosis by contamination of DNA, in particular, from carryover contamination with previously amplified DNA or extracted DNA from field samples. In this study, an uracil DNA glycosylase (UNG)-based direct PCR (udPCR) without DNA extraction process and DNA carryover contamination was developed and evaluated on PCV2 culture and field pig samples. The sensitivity of the udPCR combined with dPCR and uPCR was same or better than that of the commercial PCR (cPCR) kit (Median diagnostics, Korea) on PCV2-positive serum, lymph node and lung samples of the pigs. In addition, the udPCR method confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PCV2 DNA from previous udPCR. In clinical application, 170 pig samples (86 tissues and 84 serum) were analysed by cPCR kit and resulted in 37% (63/170) of positive reaction, while the udPCR was able to detect the PCV2 DNA in 45.3% (77/170) with higher sensitivity than cPCR. In conclusion, the udPCR developed in the study is a time, labor and cost saving method for the detection of PCV2 and providing a preventing effect for DNA carryover contamination that can occurred in PCR process. Therefore, the udPCR assay could be an useful alternative method for the diagnosis of PCV2 in the swine disease diagnostic laboratories.

• 한국식품과학회지
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• 제35권4호
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• pp.591-597
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• 2003

국내에서 시판되는 감자와 수입 감자스낵류로부터 상용 DNA 추출 kit 및 CTAB-phenol/chloroform 추출법등을 이용하여 시료특성에 따른 genomic DNA를 추출방법을 선정하고 PCR 정성검사를 실시하였다. 생감자의 경우 STE 용액으로 과량의 전분을 제거한 다음 DNA를 추출한 경우 순도 높은 DNA를 추출할 수 있었으며 상용 추출 kit를 이용한 경우 lysis buffer와 함께 ${\alpha}-/{\beta}$-amylase를 각각 또는 혼합으로 처리하거나 추출된 DNA 용액에 마지막 단계에서 효소를 처리한 시료군에서 고순도의 DNA를 추출할 수 있었으며, 효소 처리군에서는 ${\alpha}-/{\beta}$-amylase를 혼합으로 처리한 경우에 DNA 추출수율이 높았다. 냉동가공감자의 경우 silica-coated bead법을 이용하여 효소를 처리한 경우와 CTAB-페놀 클로로포름 처리군에서 DNA가 추출되었다. 또한, 각 방법으로 추출한 DNA에 대하여 감자의 내인성 유전자인 Pss 프라이머를 사용하여 PCR을 한 결과 모든 시료에서 추출된 DNA에 대하여 내부표준유전자 증폭산물이 검출되었다. 고도의 가공처리를 거친 수입 감자스낵(fabricated potato chips)과 냉동가공 감자(frozen fried potato) 등은 계면활성제인 CTAB(cetyl trimethyl ammonium bromide)과 페놀-클로로포름 혼합액을 이용하여 추출하고 이를 template로 하여 PCR 증폭을 실시하였다. 그 결과, Fig. 8에 제시한 바대로 감자의 내인성 유전자인 Pss 특이적 산물인 216bp의 산물이 냉동감자가공품과 감자칩에서 검출되었으며 재조합유전자인 New leaf plus 유래의 증폭산물(234bp)와 New lear Y유래의 증폭산물(225bp)는 검출되지 않았다. 본 실험의 결과 시료의 가공특성과 적용한 추출 kit 및 방법에 따라 genomic DNA 순도 및 추출수율이 크게 차이가 났으며 이것이 결국 PCR 결과에 의음성 혹은 의양성 등에 영향을 미치게 될 것으로 판단된다. 또한, 동일한 DNA추출방법에 의해서도 DNA가 추출되지 않은 경우가 있어서 동일한 시료에서 2회 반복 추출하는 것이 의음성결과를 피할 수 있는 방법으로 판단된다.

• 산업기술연구
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• 제28권A호
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• pp.141-147
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• 2008

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.