• 미생물학회지
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• 제44권4호
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• pp.346-351
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• 2008

본 연구에서는 임신진단등의 간편 현장진단(point of care test, POCT)에 주로 사용되고 있는 멤브레인 측면흐름 분석기법을 사용하여 인유두종 바이러스(Human papillomavirus, HPV)의 특정 서열을 검출할 수 있는 DNA array를 개발하였다. HPV type 6, 11, 16, 18, 31, 45에 특이적인 DNA 탐침들을 측면흐름 분석용 멤브레인 표면에 고정하고, biotin이 label된 MY09/11 primer를 사용하여 얻어진 HPV PCR 반응 결과물과 탐침 사이에 hybridization 반응을 유도하였다. 이후 streptavidin이 label된 colloidal gold가 교잡물의 biotin과 반응함으로써 DNA hybridization 결과를 육안으로 확인할 수 있었다. 본 연구를 통해 개발된 HPV DNA lateral flow membrane array는 기존의 HPV DNA chip 기법과 비교하여 경제적이고 편리하게 주요 HPV type을 확인할 수 있음을 보여주었다.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.123-129
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• 2021

Polyhydroxyalkanoate (PHA) synthase is a key enzyme for PHA production in microorganisms. The class IV PHA synthase is composed of two subunits: PhaC and PhaR. The PhaR subunit, which encodes the phaR gene, is only present in class IV PHA synthases. Therefore, the phaR gene is used as a biomarker for bacteria that contain a class IV PHA synthase, such as some Bacillus spp. The phaR gene was developed to screen phaR-containing Bacillus spp. The phaR screening method involved two steps: phaR gene amplification by PCR and phaR amplicon detection using a DNA lateral flow assay. The screening method has a high specificity for phaR-containing Bacillus spp. The lowest amount of genomic DNA of B. thuringiensis ATCC 10792 that the phaR screening method could detect was 10 pg. This novel screening method improves the specificity and sensitivity of phaR gene screening and reduces the time and cost of the screening process, which could enhance the opportunity to discover good candidate PHA producers. Nevertheless, the screening method can certainly be used as a tool to screen phaR-containing Bacillus spp. from environmental samples.

• 미생물학회지
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• 제39권2호
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• pp.114-117
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• 2003

유전자의 기능을 분석하는 과정에서 특정한 염기 서열의 존재여부를 확인하는 분석법은 필수적이다. 현재 사용되고 있는 Southern및 Northern blotting방법은 시간이 오래 걸리며, 온도 등과 같은 외부 조건을 엄격하게 조절하여야 한다. 본 연구에서는 측방유동방식을 이용한 크로마토그라피법을 응용하여 새로운 간편용 DNA분석법을 개발하였다. 이 측방유동형 DNA 분석 스트립은 시료가 적용되는 샘플패드, 이동하여 분리되고 교잡반응이 일어나는 전개용 막, 그리고 시료가 계속하여 이동하기 위한 흡수패드로 구성되어 있다. 모델 시스템으로 HIV와 HCV에 대한 포획 및 표적 DNA를 합성하고 스트립을 제조하였다. 시료를 샘플패드에 적하한 후 교잡반응체의 생성여부와 상대적인 양은 GSI형광 스캐너로 분석하였다. 교잡반응이 매우 빠르게 진행되고 세척과정이 없음에도 불구하고 비특이적인 교차 반응이 거의 관찰되지 않았다. 기존의 DNA 교잡방법과 비교하여 볼 때 이 새로운 방법으로 DNA/DNA 교잡 실험을 보다 더 쉽고, 간편하고, 그리고 빠르게 할 수가 있을 것으로 예상된다.

• Parasites, Hosts and Diseases
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• 제59권2호
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• pp.167-171
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• 2021

Haemonchosis remains a significant problem in small ruminants. In this study, the assay of recombinase polymerase amplification (RPA) combined with the lateral flow strip (LFS-RPA) was established for the rapid detection of Haemonchus contortus in goat feces. The assay used primers and a probe targeting a specific sequence in the ITS-2 gene. We compared the performance of the LFS-RPA assay to a PCR assay. The LFS-RPA had a detection limit of 10 fg DNA, which was 10 times less compared to the lowest detection limit obtained by PCR. Out of 24 goat fecal samples, LFS-RPA assay detected H. contortus DNA with 95.8% sensitivity, compared to PCR, 79.1% sensitivity. LFS-RPA assay did not detect DNA from other related helminth species and demonstrated an adequate tolerance to inhibitors present in the goat feces. Taken together, our results suggest that LFS-RPA assay had a high diagnostic accuracy for the rapid detection of H. contortus and merits further evaluation.

• International Journal of Oral Biology
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• 제44권1호
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• pp.8-13
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• 2019

Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to $3{\mu}M$ or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, $2{\mu}M$ of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.

• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1672-1683
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• 2021

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The bla_CARB-17 gene is an intrinsic β-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this bla_CARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg²⁺, 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63℃ for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 × 10^-4 ng/μl, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

• 대한두경부종양학회지
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• 제12권2호
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• pp.153-160
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• 1996

Regardless of the prognostic factors in papillary thyroid cancer, such as sex, age, size of tumor, extent of disease, and distant metastasis, the prognosis of papillary thyroid cancer is sometimes difficult to predict from clinical and microscopic analysis alone and additional prognostic indicators are needed. Recent studies of thyroid cancer have indicated that DNA aneuploidy may be correlated to the biological behavior of malignancy and inversely correlated to the prognosis, but it still remains contraversal. We performed this study to assess DNA ploidy patterns in relation with the previously known prognostic factors in AMES scoring system and lateral neck node metastasis in papillary thyroid cancer. A series of 132 patients with papillary thyroid cancer and 80 patients with benign thyroid tumor(27 follicular adenomas and 53 adenomatous goiters) as a control group from October 1993 to Feburary 1995 were analyzed and their nuclear DNA content was measured with flow cytometry using fresh tissue specimens. DNA aneuploidy was found in 8(6.1%) in papillary cancer and 8(10%) in benign tumor. S-phase traction(SFP) and proliferative index(PI) were higher in thyroid cancers, being 2.18$\pm$4.24%, 6.34$\pm$4.94% in the papillary thyroid cancers and 1.97$\pm$2.93%, 4.44$\pm$3.80% in the benign tumors, respectively. However there was no significant difference of values between two groups(p>0.05). Among variable prognostic factors studied(age, sex, size of tun or, extent of disease, distant metastasis in AMES scoring system and lateral neck node metastasis), DNA aneuploidy was found to be common in distant metastasis(p<0.001) and in lateral neck node metastasis(p>0.035), but there was no significant difference between the high risk and low risk group according to the AMES scoring system(p<0.08). In our study, DNA aneuploidy was not valuable in determining the presence of malignancy and did not correlate to the AMES scoring system. However, follow-up study of more cases will be needed for accurate information about the DNA ploidy as a independent prognostic factor.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.454-464
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• 2019

Salmonellosis is a highly contagious bacterial disease that threatens both human and poultry health. Tests that can detect Salmonella in the field are urgently required to facilitate disease control and for epidemiological investigations. Here, we combined loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) to rapidly and accurately detect Salmonella. LAMP primers were designed to target the Salmonella invA gene. LAMP conditions were optimized by adjusting the ratio of inner to outer primers, $MgSO_4$ concentration, dNTP mix concentration, amplification temperature, and amplification time. We evaluated the specificity of our novel LAMP-LFD method using six Salmonella species and six related non-Salmonella strains. All six of the Salmonella strains, but none of the non-Salmonella strains, were amplified. LAMP-LFD was sensitive enough to detect concentrations of Salmonella enterica subsp. enterica serovar Pullorum genomic DNA as low as $89fg/{\mu}l$, which is 1,000 times more sensitive than conventional PCR. When artificially contaminated feed samples were analyzed, LAMP-LFD was also more sensitive than PCR. Finally, LAMP-LFD gave no false positives across 350 chicken anal swabs. Therefore, our novel LAMP-LFD assay was highly sensitive, specific, convenient, and fast, making it a valuable tool for the early diagnosis and monitoring of Salmonella infection in chickens.

• 대한기계학회논문집B
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• 제35권12호
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• pp.1309-1316
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• 2011

본 논문에서는 올리고-dT 자성입자와 측면방향 자기영동 기술을 기반으로 하는 초고속 RNA 추출칩을 소개한다. 센자성 와이어에 유도된 고구배자장에 의해 RNA가 결합된 올리고-dT 자성입자를 분리함으로써 용해된 혈액으로부터 고속으로 RNA를 추출하였다. 유속이 20 ml/h까지 자성입자를 80% 이상의 효율로 분리할 수 있었으며, 분리시간은 총 1분 이내였다. 추출된 시료로부터 단백질에 대한 RNA 흡광비율(absorbance ratio of RNA to protein: A260/A280)이 1.7 이상임을 확인하였고, 따라서 추출된 RNA가 매우 순수함을 보였다. 추출된 RNA를 사용하여 cDNA 합성과 PCR을 수행하였으며, 이로부터 개발된 초고속 RNA 추출칩이 적은 양의 시료만으로 간편하며 빠르고 정교한 RT-PCR을 수행하는데 실용적임을 확인하였다.

• Animal Bioscience
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• 제36권4호
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• pp.654-670
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• 2023

Objective: This study aimed to develop loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and compare it with LAMP-AGE, polymerase chain reaction (PCR), and standard Salmonella culture as reference methods for detecting Salmonella contamination in animal products and animal production environmental samples. Methods: The SalInvA01 primer, derived from the InvA gene and designed as a new probe for LFD detection, was used in developing this study. Adjusting for optimal conditions by temperature, time, and reagent concentration includes evaluating the specificity and limit of detection. The sampling of 120 animal product samples and 350 animal production environmental samples was determined by LAMP-LFD, comparing LAMP-AGE, PCR, and the culture method. Results: Salmonella was amplified using optimal conditions for the LAMP reaction and a DNA probe for LFD at 63℃ for 60 minutes. The specificity test revealed no cross-reactivity with other microorganisms. The limit of detection of LAMP-LFD in pure culture was 3×10² CFU/mL (6 CFU/reaction) and 9.01 pg/μL in genomic DNA. The limit of detection of the LAMP-LFD using artificially inoculated in minced chicken samples with 5 hours of pre-enrichment was 3.4×10⁴ CFU/mL (680 CFU/reaction). For 120 animal product samples, Salmonella was detected by the culture method, LAMP-LFD, LAMP-AGE, and PCR in 10/120 (8.3%). In three hundred fifty animal production environmental samples, Salmonella was detected in 91/350 (26%) by the culture method, equivalent to the detection rates of LAMP-LFD and LAMP-AGE, while PCR achieved 86/350 (24.6%). When comparing sensitivity, specificity, positive predictive value, and accuracy, LAMP-LFD showed the best results at 100%, 95.7%, 86.3%, and 96.6%, respectively. For Kappa index of LAMP-LFD, indicated nearly perfect agreement with culture method. Conclusion: The LAMP-LFD Salmonella detection, which used InvA gene, was highly specific, sensitive, and convenient for identifying Salmonella. Furthermore, this method could be used for Salmonella monitoring and primary screening in animal products and animal production environmental samples.