• Asian Pacific Journal of Cancer Prevention
• /
• 제13권7호
• /
• pp.3455-3460
• /
• 2012

Objectives: To report the prevalence of atypical squamous cells of undetermined significance and atypical squamous cells-cannot exclude high squamous intraepithelial lesions and to determine the possible association of Pap test results with high-risk human papillomavirus and high squamous intraepithelial lesions in women from Duhok, Iraq. Design: A prospective, observational study was conducted between January 2005 and December 2011. Overall, 596 women with a cervicovaginal Pap test showing atypical squamous cells of undetermined significance and 93 atypical squamous cells-cannot exclude high squamous intraepithelial lesion for whom pathologic follow-up was available were studied. Follow-up consisted of repeat cytology, colposcopy and histology. High risk human papillomavirus DNA testing was performed on exfoliated cervical cells from 106 women, using conventional PCR after at least 36 months from the initial Pap smear. Results: Significantly high proportions of both atypical squamous cells of undetermined significance (87.9%) and atypical squamous cells-cannot exclude high squamous intraepithelial lesion (62.4%) demonstrated no significant lesion on subsequent follow up. Low squamous intraepithelial lesions were observed in 1.7% of cases of atypical squamous cells of undetermined significance and in 5.4% of atypical squamous cells-cannot exclude high squamous intraepithelial lesion. High squamous intraepithelial lesion was demonstrated in 0.8% and 16.1% respectively. In the latter there was also one case of invasive carcinoma. High-risk HPV DNA was demonstrated in 40% of atypical squamous cells of undetermined significance and 57.1% of atypical squamous cells-cannot exclude high squamous intraepithelial lesions. Conclusions: Since both atypical squamous cells of undetermined significance and atypical squamous cells-cannot exclude high squamous intraepithelial lesion identify patients who are at an increased risk for the development of high squamous intraepithelial lesions and a considerable percentage harbor high risk-HPV, both should be retained as diagnostic categories and patients warrant a diligent follow up and testing for high risk-HPV DNA. Colposcopic evaluation and biopsy, when indicated, are a must.

• Journal of Genetic Medicine
• /
• 제12권2호
• /
• pp.72-78
• /
• 2015

Recently, noninvasive prenatal test (NIPT) has been adopted as a primary screening tool for fetal chromosomal aneuploidy. The principle of NIPT lies in isolating the fetal fraction of cell-free DNA in maternal plasma and analyzing it with bioinformatic tools to measure the amount of gene from the target chromosome, such as chromosomes 21, 18, and 13. NIPT will contribute to decreasing the need for unnecessary invasive procedures, including amniocentesis and chorionic villi sampling, for confirming fetal aneuploidy because of its higher positive predictive value than that of the conventional prenatal screening method. However, its greater cost than that of the current antenatal screening protocol may be an obstacle to the adoption of this innovative technique in clinical practice. Digital polymerase chain reaction (dPCR) is a novel approach for detecting and quantifying nucleic acid. dPCR provides real-time diagnostic advantages with higher sensitivity, accuracy, and absolute quantification than conventional quantitative PCR. Since the groundbreaking discovery that fetal cell-free nucleic acid exists in maternal plasma was reported, dPCR has been used for the quantification of fetal DNA and for screening for fetal aneuploidy. It has been suggested that dPCR will decrease the cost by targeting specific sequences in the target chromosome, and dPCR-based noninvasive testing will facilitate progress toward the implementation of a noninvasive approach for screening for trisomy 21, 18, and 13. In this review, we highlight the principle of dPCR and discuss its future implications in clinical practice.

• Journal of Genetic Medicine
• /
• 제8권1호
• /
• pp.1-16
• /
• 2011

현재 사용되고 있는 침습적 산전진단법(양수천자, 융모막샘플링)은 1-2%의 태아 손실이 초래되어, 비침습적 산전진단법이 산전진단의 궁극적인 목표로 대두되어 왔다. 1997년 Dr. Lo에 의해서 임신부 혈장 내에 세포 유리 태아 DNA (cffDNA)의 존재가 발견된 후 비침습적 산전진단의 새로운 가능성이 열렸으며, 과거 10년간 이에 대한 연구의 많은 진전을 보여주고 있다. 최근에 cffDNA를 이용한 Hemophilia A와 듀센형 근이영양증 등 반성 유전병(X-linked disorders) 진단에 필수적인 산전태아의 성 판정과 RhD-음성 임신부에서 태아의 RhD유전자 핵형 분석 등이 이미 외국에서 임상적으로 적용되고 있으나, 한국에서는 아직 실용화되지 않고 있다. CffDNA의 임상 사용에는 여전히 많은 제약점이 있으며, 이는 임신부 혈장 내 cffDNA 양에 비해 많은 양의 모태 DNA가 존재하고, 종래에 사용되었던 특이적인 Y염색체 유전자(Y-specific gene)는 남아 태아 임신 시에만 적용된다는 것에 기인한 다. 따라서 모든 태아에 적용할 수 있는 태아 성과 무관한 마커(sex-independent universal fetal marker as internal positive controls)가 요구되며, 이를 이용하여 정확한 태아 DNA를 검출할 수 있다. 본 연구진은 국내 처음으로 임신부 혈장 내에 cffDNA를 이용하여 SRY 유전자, RhD-exon 7, 태아 성과 무관한 DNA마커(universal fetal DNA marker)로써 RASSF1A 유전자를 실시간 중합효소연쇄반응(RT- PCR)을 사용하여 뛰어난 결과를 얻었다. 이는 한국에서 처음으로 성공적으로 시도된 것이다. 연구결과에서 산전 태아 성 판별과 산후 태아의 성이 100% 일치하였으며, 임신 주기별 SRY 수치는 임신이 진행할수록 증가함을 확인할 수 있었다. 따라서 이러한 방법은 혈우병 A, 듀센형 근이영양증, 선천성 부신증식증과 연골 무형성증의 진단과 치료 상담에 이용할 수 있으며 50%에서 침습적인 방법을 줄일 수가 있다. 또한, RhD-음성 임신부 대상으로 태아의 성 판정과 RhD 태아 유전자형을 분석한 결과 RhD-음성 태아를 정확히 검출함으로써 앞으로 기존 양수천자 등 침습적 검사를 대체할 수 있을 것이다. 특히 이는 치료가 필요 없는 RhD-음성 태아에서 RhD-면역글로불린의 예방적 치료를 사전에 막을 수 있어, 임신부 건강을 보호하고 의료 비용을 줄일 수 있는 큰 장점을 가진다. 한국에서 최초로 시도된 임신부 혈장 내 cffDNA를 이용한 본 연구의 성공은 비침습적 산전진단 임상 적용의 새 길을 제시하였다. 따라서 이를 각 유전질환의 산전진단에 유용하게 활용하는 것은 태아와 임신부의 건강 증진과 의료비용 절약 등 개인과 국가에 많은 기여를 할 것으로 사료된다.

• 한국동물위생학회지
• /
• 제23권4호
• /
• pp.349-360
• /
• 2000

An epidemiological survey was conducted to investigate fowl typhoid outbreaks in Kyungnam province of Korea. The causative agent, salmonella gallinarum was isolated from 68 chicken samples of tentatively diagnosed fowl typhoid cases occurred during the period from January 1996 to September 1999. Comparative studies were also carried out to evaluate the diagnostic methods for detection of S gallinam The results obtained were as follows; 1. Of the 68 cases of tentatively diagnosed fowl typhoid, 56 (82%) cases were determined as fowl typhoid by biochemical test and pathological findings. The other 12 (18%) cases were determined as paratyphoid. 2. Fowl typhoid outbreaks occur continuously all seasons in the year, however the incidence was remarkably increased from May to September. 3. The frequency of incidence of fowl typhoid in terms of regional distribution was relatively high in egg-laying hens facilities, and the mode of transmission is likely to be either egg-to-egg or lateral transfer by wild birds or rats. 4. All of 18 isolates from 56 cases were identified as S gallinarum by biochemical and serological test. 5. Antimicrobial drug susceptibility test against 18 isolates showed that the isolates were highly susceptible to ASH, CZ, CF and GM (above 90%), whereas those strains were 100% resistant to EM, NA and PC. 6. S gallinarum rfbS gene was targeted to be amplified by PCR for comparative detection of S gallinarum in the experimentally infected chickens. The amplified 720bp DNA fragment, which is specific in D serogroup strains of S enterica subspecies was confirmed by agarose gel electrophoresis. 7. A comparison made between fecal culture and PCR-method revealed that later-method was relatively higher in detection rate than that of former method for S gallinarum. 8. Comparison of currently applied methods, rapid serum agglutination test (RST) and microplate agglutination test (MAT), with experimentally infected chickens were made to evaluate sensitivity of detection by neutralizing antibody titration. Both methods detected neutralizing antibodies from the challenged chickens of 5 day post infection. However, positive reactions were determined after 7 and 9 days post infection by MAT and RST, respectively.

• 대한임상검사과학회지
• /
• 제47권1호
• /
• pp.17-23
• /
• 2015

A fine needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant in thyroid nodules. However, between 10 and 30% of the FNABs of thyroid nodules are diagnosed as 'indeterminate'. A molecular method is needed to reduce unnecessary surgery in this group. In Korea, most thyroid cancer is classic papillary type and BRAFV600E mutation is highly prevalent. Thus, this study compared the pyrosequencing method with the conventional direct DNA sequencing and PCR-RFLP analysis and investigated the evaluation of preoperative BRAFV600E mutation analysis as an adjunct diagnostic method with routine FNABs. Sixty-five (78.3%) of 83 histopathologically diagnosed malignant nodule revealed positive BRAFV600E mutation on pyrosequencing analysis. In detail, 65 (83.8%) of 78 papillary thyroid carcinomas sample showed positive BRAFV600E mutation. None of 29 benign nodules had in pyrodequencing, direct DNA sequencing and PCR-RFLP. Out of 31 thyroid nodules classified as 'indeterminate' on cytological examination preoperatively, 28 cases turned out to be malignant: 24 papillary thyroid carcinomas. Among that, 16 (66.7%) classic papillary thyroid carcinomas had BRAFV600E mutation. Among 65 papillary thyroid carcinomas with positive BRAFV600E mutation detected by pyrosequencing analysis, each 3 cases and 5 cases did not show BRAFV600E mutation by direct DNA sequencing and PCR-RFLP analysis. Therefore, pyrosequencing was superior to direct DNA sequencing and PCR-RFLP in detecting the BRAFV600E mutation of thyroid nodules (p =0.027). Detecting BRAFV600E mutation by pyrosequencing was more sensitivity, faster than direct DNA sequencing or PCR-RFLP.

• 한국간호교육학회지
• /
• 제19권4호
• /
• pp.675-683
• /
• 2013

Purpose: Nineteen articles were analyzed to gather opinions and nursing implications about NIPT recently launched. Methods: Nineteen articles were selected from EBSCO (eBook business collection), Google Scholar, and two Korean academic d-bases with key words 'prenatal screening testing', 'prenatal genetic diagnostic testing', NIPT or 'cell free DNA (cfDNA)'. Authors developed a framework for analyzing the 19 articles including opinions and suggestions for future implications. Results: Eleven articles written by the first author affiliated with medicine or genetics, viewed NIPT as promising because of safety, accuracy, early detectability and cost effectiveness. Articles written by journalists or authors affiliated with history and ethics were concerned with the possible risk of ELSI (ethical, legal, social issues), erratic interpretation of test results, and lack of genetic counseling service. Conclusion: With consideration of Korean clinical, and legal circumstances, not only pregnant women and families but also health professionals must prepare for clinical NIPT implications including updating prenatal genetic testing, counseling services, protecting ELSI and amulticultural team approach.

• International Journal of Oral Biology
• /
• 제48권2호
• /
• pp.9-18
• /
• 2023

The rapid detection of bacteria in the oral cavity, its species identification, and bacterial count determination are important to diagnose oral diseases caused by pathogenic bacteria. The existing clinical microbial diagnosis methods are time-consuming as they involve observing patients' samples under a microscope or culturing and confirming bacteria using polymerase chain reaction (PCR) kits, making the process complex. Therefore, it is required to analyze the development status of substances and systems that can rapidly detect and analyze pathogenic microorganisms in the oral cavity. With research advancements, a close relationship between oral and systemic diseases has been identified, making it crucial to identify the changes in the oral cavity bacterial composition. Additionally, an early and accurate diagnosis is essential for better prognosis in periodontal disease. However, most periodontal disease-causing pathogens are anaerobic bacteria, which are difficult to identify using conventional bacterial culture methods. Further, the existing PCR method takes a long time to detect and involves complicated stages. Therefore, to address these challenges, the concept of point-of-care (PoC) has emerged, leading to the study and implementation of various chair-side test methods. This study aims to investigate the different PoC diagnostic methods introduced thus far for identifying pathogenic microorganisms in the oral cavity. These are classified into three categories: 1) microbiological tests, 2) microchemical tests, and 3) genetic tests. The microbiological tests are used to determine the presence or absence of representative causative bacteria of periodontal diseases, such as A. actinomycetemcomitans, P. gingivalis, P. intermedia, and T. denticola. However, the quantitative analysis remains impossible, and detecting pathogens other than the specific ones is challenging. The microchemical tests determine the activity of inflammation or disease by measuring the levels of biomarkers present in the oral cavity. Although this diagnostic method is based on increase in the specific biomarkers proportional to inflammation or disease progression in the oral cavity, its commercialization is limited due to low sensitivity and specificity. The genetic tests are based on the concept that differences in disease vulnerability and treatment response are caused by the patient's DNA predisposition. Specifically, the IL-1 gene is used in such tests. PoC diagnostic methods developed to date serve as supplementary diagnostic methods and tools for patient education, in addition to existing diagnostic methods, although they have limitations in diagnosing oral diseases alone. Research on various PoC test methods that can analyze and manage the oral cavity bacterial composition is expected to become more active, aligning with the shift from treatment-oriented to prevention-oriented approaches in healthcare.

• Journal of Genetic Medicine
• /
• 제6권2호
• /
• pp.146-154
• /
• 2009

Multiplex ligation dependent probe amplification (MLPA)은 탐침자를 표적지에 교잡시킨 후, ligation 시키고, 그 산물을 중합효소연쇄반응으로 증폭시킴으로써 표적지의 존재여부 또는 농도를 확인할 수 있는 방법으로, 그 원리가 소개된 이래로 여러 유전자들에 대한 거대결실 및 중복돌연변이에 대한 탐색에 이용되었다. 유전자진단은 질환에 관련된 유전자에 대한 돌연변이를 탐색함으로써 질환을 진단하는 방법으로, 단일유전자 결핍 질환에 대한 유전자진단은 주로 중합효소연쇄반응과 염기서열 분석 방법을 통한 점돌연변이의 탐색에 집중되어 있다. 거대결실 또는 중복돌연변이의 경우, 특히 이형접합자를 형성하게 되는 경우는 중합효소 연쇄반응을 통하여 결실 또는 중복돌연변이 여부의 확인이 힘들다. PCR 방법에 기초하여 유전자의 농도(gene dosage)를 알 수 있는 방법으로 MLPA 방법이 소개되면서 거대결실 또는중복돌연변이를 포함하고 있던 질병 관련돌연변이들의 규명이 한층 쉬워졌다. MLPA의 원리를 응용하여 단순한 유전자의 농도 측정뿐 아니라 유전자내의 메칠화양상의 차이를 확인하거나, 염색체의 배수체 이상 등 염색체이상의 돌연변이 규명과, 전체 유전자의 크기가 비교적 커서 거대결실 돌연변이를 많이 동반하는, 주로 우성유전의 암 관련 유전자 돌연변이의 규명에 유용하게 이용된다. MLPA는 상용적인 중합효소연쇄반응으로 확인할 수 없는 유전자의 농도를 효과적으로 규명할 수 있는 방법으로, 적은 양의 주형 DNA만을 사용하고, 한가지의 실험원리로 다양한 응용이 가능하며 high-throughput이 가능한 장점을 가지는 반면, 주형 DNA의 질에 결과의 의존도가 높고, 민족 또는 개인간의 차이를 보일 수 있는 표적 DNA 염기서열 내의 single nucleotide polymorphism (SNP) 등으로 인해 분석의 오류가 생길 수 있으며, 양적 차이를 규명하는 것이므로 수 차례의 대조군 검사가 함께 진행되어야 하는 단점이 있다. 여기서는 MLPA를 이용하여 질병유전자의 돌연변이를 밝힌 사례를 바탕으로 MLPA의 원리와 탐색할 수 있는 돌연변이의 종류, 그리고 이 방법의 장단점에 대해 고찰해 보고자 한다.

• Parasites, Hosts and Diseases
• /
• 제52권2호
• /
• pp.143-149
• /
• 2014

To evaluate the seroprevalence against circumsporozoite protein (CSP) of Plasmodium vivax in sera of Korean patients, the central repeating domain (CRD) of CSP was cloned and analyzed. From the genomic DNA of patient's blood, 2 kinds of CSPs were identified to belong to a VK210 type, which is the dominant repeating of GDRA(D/A)GQPA, and named as PvCSPA and PvCSPB. Recombinantly expressed his-tagged PvCSPA or PvCSPB in Escherichia coli reacted well against sera of patients in western blot, with the detecting rate of 47.9% (58/121), which included 15 cases positive for PvCSPA, 6 cases positive for PvCSPB, and 37 cases for both. The mixture of PvCSPA and PvCSPB was loaded to a rapid diagnostic test kit (RDT) and applied with the same set of patient sera, which resulted in detection rates of 57.0% (69/121). When the protein sequences of PvCSPA were compared with those of P. vivax in endemic regions of India and Uganda, they were compatibly homologous to PvCSPA with minor mutations. These results suggested that the recombinant PvCSPA and PvCSPB loaded RDT may be a milestone in latent diagnosis which has been a hot issue of domestic malaria and important for radical therapy in overlapped infections with P. falciparum in tropical and subtropical areas. During the biological process of malarial infection, exposure of CSP to antigen-antibody reaction up to 57.0% is the first report in Korea.

• 농업과학연구
• /
• 제47권3호
• /
• pp.673-681
• /
• 2020

Human enteric Adenovirus 41 (HueAdV-41) is a major waterborne virus that causes human gastroenteritis and is classified as a viral group I double-strand DNA virus, Adenoviridae. HueAdV-41 has been detected with the polymerase chain reaction (PCR) in various samples such as ground water. However, the PCR-based diagnostic method has problems such as reaction time, sensitivity, and specificity. Thus, the loop-mediated isothermal amplification (LAMP) assay has emerged as an excellent method for field applications. In this study, we developed a LAMP system that can rapidly detect HueAdV-41 with high specificity and sensitivity. HueAdV-41 specific LAMP primer sets were tested through a specific, non-specific selection and sensitivity test for three prepared LAMP primer sets, of which only one primer set and optimum reaction temperature were selected. The developed LAMP primer set condition was confirmed as 63℃, and the sensitivity was 1 copy. In addition, to confirm the system, a LAMP positive reaction was developed with the restriction enzyme Taq I (T/GCC). The developed method in this study was more specific, rapid (typically within 2 - 3 hours), and highly sensitive than that of the conventional PCR method. To evaluate and verify the developed LAMP assay, an artificial infection test was done with five cDNAs from groundwater samples, and the results were compared to those of the conventional PCR method. We expect the developed LAMP primer set will be used to diagnose HueAdV-41 from various samples.