• Journal of Microbiology and Biotechnology
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• 제22권9호
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• pp.1301-1306
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• 2012

We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 non-respiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was ${\leq}35$ and the ratio of real-time RT-PCR and real-time PCR load was ${\geq}1.51$. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including non-respiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.

• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.120-129
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• 2016

An industrial complex in Wonju, contaminated with trichloroethene (TCE), was one of the most problematic sites in Korea. Despite repeated remedial trials for decades, chlorinated ethenes remained as sources of down-gradient groundwater contamination. Recent efforts were being made to remove the contaminants of the area, but knowledge of the indigenous microbial communities and their dechlorination abilities were unknown. Thus, the objectives of the present study were (i) to evaluate the dechlorination abilities of indigenous microbes at the contaminated site, (ii) to characterize which microbes and reductive dehalogenase genes were responsible for the dechlorination reactions, and (iii) to develop a PCE-to-ethene dechlorinating microbial consortium. An enrichment culture that dechlorinates PCE to ethene was obtained from Wonju stream, nearby a trichloroethene (TCE)-contaminated industrial complex. The community profiling revealed that known organohalide-respiring microbes, such as Geobacter, Desulfuromonas, and Dehalococcoides grew during the incubation with chlorinated ethenes. Although Chloroflexi populations (i.e., Longilinea and Bellilinea) were the most enriched in the sediment microcosms, those were not found in the transfer cultures. Based upon the results from pyrosequencing of 16S rRNA gene amplicons and qPCR using TaqMan chemistry, close relatives of Dehalococcoides mccartyi strains FL2 and GT seemed to be dominant and responsible for the complete detoxification of chlorinated ethenes in the transfer cultures. This study also demonstrated that the contaminated site harbors indigenous microbes that can convert PCE to ethene, and the developed consortium can be an important resource for future bioremediation efforts.

• Asian Pacific Journal of Cancer Prevention
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• 제17권11호
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• pp.4917-4920
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• 2016

For advanced non-small-cell lung cancer (NSCLC) cases, a platinum-based regimen is the first-line chemotherapy treatment. The excision repair cross-complementing group 1 (ERCC1) plays an important role in DNA repair and has been related to resistance to platinum chemotherapy. This study aimed to investigate the effects of the ERCC1 (C118T) polymorphism on treatment response in 26 Thai advanced NSCLC patients receiving first line platinum-based chemotherapy during January to July 2015 at King Chulalongkorn Memorial Hospital (KCMH). DNA was extracted from peripheral blood lymphocytes and the single nucleotide polymorphism of ERCC1 was genotyped using a real-time PCR method with the TaqMan assay. The distribution of C/C, C/T and T/T genotypes was 57.7 %, 34.6 % and 7.7 %, respectively. The response rate to platinum-based chemotherapy in the wild type (C/C) of ERCC1 (C118T) was better than with the variant types (C/T and T/T) but the difference was not statistically significant (29.7% vs 9.1%, P=0.274). The results showed that a genetic polymorphism in ERCC1 might influence patient response to platinum-based chemotherapy. Further multicenter studies are now required to confirm the results of our study.

• Asian Pacific Journal of Cancer Prevention
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• 제14권1호
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• pp.299-302
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• 2013

Methylenetetrahydrofolate reductase (MTHFR) has been reported to be associated with DNA methylation, an epigenetic feature frequently found in gastric cancer. We conducted a case-control study to explore the association of MTHFR C677T polymorphisms with gastric cancer risk and its relation with the DNA methylation of COX-2, MGMT, and hMLH1 genes. Genotyping of P16, MGMT and HMLH1 was determined by methylation-specific PCR after sodium bisulfate modification of DNA, and genotyping of MTHFR C677T was conducted by TaqMan assays using the ABI Prism 7911HT Sequence Detection System. Folate intake was calculated with the aid of a questionnaire. Compared with the MTHFR 677CC genotype, the TT genotype was significantly associated with 2.08 fold risk of gastric cancer when adjusting for potential risk factors. Individuals who had an intake of folate above $310{\mu}g$/day showed protective effects against gastric cancer risk. The effect of MTHFR C677T polymorphisms on the risk of gastric cancer was modified by folate intake and methylation status of MGMT (P for interaction <0.05).

• Asian Pacific Journal of Cancer Prevention
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• 제13권12호
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• pp.6187-6190
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• 2012

We aimed to investigate DNA repair gene expression of response to chemotherapy among gastric patients, and roles in the prognosis of gastric cancer. A total of 209 gastric cancer patients were included in this study between January 2007 and December 2008, all treated with chemotherapy. Polymorphisms were detected by real time PCR with TaqMan probes, and genomic DNA was extracted from peripheral blood samples. The overall response rate was 61.2%. The median progression and overall survivals were 8.5 and 18.7 months, respectively. A significant increased treatment response was found among patients with XPG C/T+T/T or XRCC1 399G/A+A/A genotypes, with the OR (95% CI) of 2.14 (1.15-4.01) and 1.75 (1.04-3.35) respectively. We found XPG C/T+T/T and XRCC1 399 G/A+A/A were associated with a longer survival among gastric cancer patients when compared with their wide type genotypes, with HRs and 95% CIs of 0.49 (0.27-0.89) and 0.56 (0.29-0.98) respectively. Selecting specific chemotherapy based on pretreatment genotyping may be an innovative strategy for further studies.

• 대한의생명과학회지
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• 제29권4호
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• pp.220-230
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• 2023

Identification of the pathogens causing infection is important in terms of patient's health management and infection control. Synovial fluids could be used as clinical samples to detect causative pathogens of prosthetic joint infections (PJIs) using molecular diagnostic assays, therefore, normalization and validation of clinical samples are necessary. Microbial culture is considered the gold standard for all infections, including PJIs. Recently, molecular diagnostic methods have been developed to overcome the limitation of microbial culture. Therefore, guideline for validating clinical samples to provide reliable results of molecular diagnostic assays for infectious diseases is required in clinical field. The present study aimed to develop an accurate validating method of synovial fluid clinical samples using GAPDH gene as an internal control to perform the quantitative PCR TaqMan probe assay to detect pathogens causing PJIs.

• 한국식품위생안전성학회지
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• 제32권3호
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• pp.199-205
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• 2017

인수공통 감염증의 하나인 톡소포자충의 검출을 위해서는 대부분은 ELISA 법이 사용 되고 있으나, 충체가 사멸된 후에도 양성반응이 나타나는 등 사용에 제한이 있다. 반면 유전자 검출법은 현재 감염상태를 확인 할 수 있기 때문에 식중독 원인조사 등에 적합하다고 판단되어 이를 활용하여 본 연구를 진행하였다. 톡소포자충의 유전정보를 통해 529 repeat region의 염기서열을 얻고, 프라이머 및 TaqMan 프로브를 설계하여 real-time PCR을 이용한 검출법을 개발하였다. 검출한계(lower limit of detection) 및 적정곡선을 확인한 결과 10 genomic DNA copy가 검출한계로 확인되었고, 정량을 위한 곡선은 $10^1{\sim}10^6$ DNA copies까지 0.999의 $R^2$ 값을 나타내었다. 개발된 검출법의 증폭효율을 비교하기 위해 B1 gene 타겟 프라이머 세트와 타입별 검출한계를 비교한 결과, type 1, 2, 3 톡소포자충에서 같거나 더 나은 검출한계를 보였다. 또한 식품에서 주로 분리되는 식중독 세균 14종 및 원충 3종에 대해 특이도를 비교한 결과, 모두 음성으로 나타났다. 개발된 검출법을 식육검체에 적용하였을 때 type 1, 2, 3에서 모두 원활한 검출결과를 보여 증폭방해물질이 존재하지 않은 것으로 확인되었다. 본 연구를 통해 개발된 유전자검출법은 국내 유통 중인 식육에서 인수 공통감염 원충의 하나인 톡소포자충의 감염 여부를 확인하는 사전적 모니터링의 방법으로 활용될 예정이다.

• Journal of Veterinary Science
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• 제22권6호
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• pp.87.1-87.12
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• 2021

Background: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10⁰ copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

• Journal of Plant Biotechnology
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• 제45권1호
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• pp.63-70
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• 2018

Brazzein은 열대식물인 P. brazzeana Baillon의 과실에서 분리된 가장 작은 감미단백질로 토착민들의 단맛원료로 사용되어 왔다. Brazzein은 sucrose보다 분자량 기준으로 500 ~ 2000배, 몰 기준으로 9500배 당도가 높아 감미료로써 매우 높은 평가를 받고 있다. 그러나 이 감미단백질은 재배가 어렵고 생산 비용이 높아서 brazzein 단백질의 이용 가능성을 높이기 위한 대체 생산 시스템으로 형질전환 식물체 육성 하고자 하였다. 본 연구에서는 brazzein 관련 유전자를 벼에 도입하기 위하여 식물형질전환용 Ti-plasmid에 $2{\times}CaMV\;35S$ 프로모터에 의해 지배되어 발현하도록 하고, 선발 마커로 bar 유전자가 삽입된 식물발현 벡터를 구축하여 A. tumefaciens EHA105에 형질전환시켜 17개의 재분화 식물체를 육성하였다. 17개 재분화 식물체는 PCR 및 RT-PCR 분석을 통하여 유전자 도입 및 발현을 확인하였으며, TaqMan PCR을 통해 single copy로 도입된 T0 세대 9개체를 선발하였다. 또한 FST 분석을 통하여 도입 유전자가 intergenic으로 삽입된 개체 5개를 선발하였다. 이들 5개체를 이용하여 western blot 분석에 의해 단백질 발현량을 분석한 결과 선발된 모든 개체에서 발현 밴드를 확인하였다. 그 중 brazzein 단백질의 발현량이 높은 개체를 TG11으로 계통화하여 후대 종자를 육성하였다. TG11 계통은 천연 감미료 brazzein을 생산하는 새로운 벼 품종을 개발하기 위한 육종 소재로 활용 가능하다고 시사된다.

• Tuberculosis and Respiratory Diseases
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• 제58권5호
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• pp.490-497
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• 2005

연구배경 : Paraoxonase는 산소유리기 제거효소의 하나로서, 유기인제 화합물의 독소제거에 있어서 중요한 역할을 한다. 본 연구에서는 한국인 남성에서 PON1 Q192R 유전자 다형성이 흡연과 관련하여 폐암발생에 미치는 영향을 조사하였다. 대상 및 방법 : 연구대상자는 조직 병리학적으로 폐암으로 새롭게 진단받은 남성 환자 335명과, 이들과 3세 이내에서 연령을 짝지은 동수의 대조군으로 하였다. 직접면접조사를 통하여 인적사항과 직업력 그리고 흡연력, 음주력 등을 조사하였다. TaqMan 실시간 중합 효소 연쇄반응을 이용하여 PON1 유전자 다형성을 확인하고, 폐암과 흡연 그리고 PON1 유전자 다형성 유형 사이의 상호 관련성을 통계적으로 분석하였다. 결 과 : 흡연과 PON1 유전자 Q/Q형이 폐암 발생 위험도를 유의하게 증가시켰다. 흡연자에서 PON1 Q/Q형인 사람이 Q/R 혹은 R/R형인 사람에 비하여 폐암에 대한 교차비(95% 신뢰구간)가 2.56(1.52 - 4.31)으로 폐암발생 위험도가 통계적으로 유의하게 증가하였다. 비흡연자이며 PON1 Q/R 혹은 R/R형인 사람을 비교군으로 하였을 때, 비흡연자이며 PON1 Q/Q형인 군, 흡연자이며 Q/R 혹은 R/R형인 군, 흡연자이며 Q/Q형군으로 이행할수록 대응비는 모든 세포유형에서 유의하게 증가하였다. 특히 흡연자이며 Q/Q형인 사람은 비흡연자이며 PON1 Q/R 혹은 R/R형인 사람에 비하여, 편평세포암종은 53.77(6.55 - 441.14)배, 선암종은 6.25(1.38 - 28.32)배, 소세포암종은 59.94(4.66 - 770.39) 배 더 잘 생기는 것으로 나타났다. 결 론 : 흡연과 PON1 Q/Q형은 폐암의 위험인자이며, 흡연과 PON1 Q/Q형은 조직학적 유형에 관계없이 폐암발생 위험도를 서로 상승적으로 증가시키는 것으로 판단된다.