• Biomedical Science Letters
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• v.22 no.1
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• pp.24-28
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• 2016

Tuberculosis (TB) remains an unsolved community health problem since identification of its causing microorganism called Mycobacterium tuberculosis (MTB) by Robert Koch in 1882. Annually, eight million TB cases are newly reported and 2~3 million patients die from TB. Pulmonary TB is highly infectious and untreated pulmonary TB patients are believed to infect >10 people in a year. The conventional methods for diagnosis of TB are chest X-ray and isolation of the causing microorganisms from patient specimens. Screening of TB is conducted with smeared sputum in slides, and TB is confirmed by identification of MTB in cultured specimens. One of the fatal pitfalls of screening detection for smeared sputum is that it is impossible to distinguish MTB and other acid-fast bacilli (AFB) because they are stained equally with Ziehl-Neelsen (ZN) stain. Culture of MTB is the most reliable method for diagnosis of TB but it takes 4~8 weeks. In this report, we suggest a fast and highly-reliable MTB detection method that distinguishes AFB in sputum samples. Purified DNA from the AFB stained slide samples offered by The Korean Institute of Tuberculosis were used to detect infected MTB in patients. PCR, real-time PCR and reverse blot hybridization assay (REBA) methods were applied to purified DNA. Conclusively, the real-time PCR method was confirmed to produce high sensitivity and we were able to further detect drug-resistant MTB with REBA.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.51-56
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• 1997

Recently, through the development of the primer extension preamplification(PEP) method which amplifies the whole genome, simultaneous multiple DNA analysis has become possible. Whole genome from each single cell can be amplified using 15 base oligonucleotide random primer. The greatest advantage of PEP-PCR is the ability to investigate several loci simultaneously and confirm results by analysing multiple aliquots for each locus. This technique led to the development of preimplantation genetic disease diagnosis using blastomere from early embryo, sperm, polar body and oocyte. In this study, we applied PEP-PCR in 20 cases of single amniocyte and 20 cases of single chorionic villus cell for the clinical application of the prenatal and preimplantational genetic diagnosis. We analysed 7 gene loci simultaneously which are 46, 47 exons related to dystrophin gene, two VNTR (variable number tandem repeat) markers using 5'dysIII, 3'CA related to dystrophin gene and DYZ1, DYZ3, DYS14 regions on chromosome Y. In all the tests, 97.5% of PEP-PCR amplifications with single cells were successful. We obtained 38/40 (95%) accuracy in gender determination through chromosome analysis comparison. Therefore, these results have significant implications for a sperm or oocyte analysis and prenatal or preimplantational genetic diagnosis.

• Parasites, Hosts and Diseases
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• v.57 no.3
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• pp.233-242
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• 2019

Detailed description of malaria in low transmission areas is crucial for elimination. The current study aimed to provide a comprehensive description for malaria transmission in Jazan, a low transmission district, southwestern Saudi Arabia. Patients at a tertiary care hospital were recruited in our study between August 2016 and September 2018. Malaria diagnosis was performed through a species-specific nested polymerase chain reaction (nested PCR), microscopy and Paramax-$3^{TM}$ rapid detection test (RDT). Malaria was detected in 30 patients by the PCR, with point prevalence of 10.9%. Of these malaria infections, 80% was imported, 26.6% was asymptomatic and 23.3% was sub-microscopic. Malaria was reported throughout the year, with February/March and September/October peaks. Infection was significantly more in males than in females (P=0.01). Likewise, infections were detected more in febrile than in non-febrile patients (P=0.01). Adult aged 15-24 years, fever and travel were identified as high-risk factors. Malaria was primarily attributed to Plasmodium falciparum mono-infections, followed by P. vivax mono-infections and lastly to falciparum/vivax mixed infections accounting 76.6%, 16.6%, and 6.6% of PCR-confirmed malaria cases, respectively. The nested PCR was superior to the smear microscopy (sensitivity 76.6%; specificity 100%) and the RDT (sensitivity 83.3%, specificity 94.2%). The overall percent agreement between microscopy and the RDT was 92.7% (kappa=0.63). High proportion of imported malaria including sub-microscopic and sub-patent cases were described. We suggest that incorporation of molecular tool into the conventional malaria diagnosis is beneficial in Jazan district.

• Korean Journal of Veterinary Service
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• v.31 no.4
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• pp.485-492
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• 2008

Salmonella enterica serovars Gallinarum(SG, causative agent of fowl typhoid) and S Pullorum(SP, causative agent of pullorum disease) are very important bacterial pathogens in poultry industry. They share some common antigenic properties though the characteristics of outbreaks are quite different. To discriminate between SG and SP, we developed two rapid diagnostic methods, allele-specific real-time PCR and 3'-tailed PCR over 2 single nucleotide polymorphisms ($237^{th}\;and\;598^{th}$). In both methods, $237^{th}$ allele was found to be a good target for differential diagnosis, while $598^{th}$ allele produced some non-specific reactions.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.1-6
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• 2007

We describe a multiplex PCR method that can detect and differentiate simultaneously four different kinds of DNA viruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis B virus (HBV) and parvovirus B19 (B19). Primers for the multiplex PCR reaction were designed to amplify specific regions of the EBV (pol), CMV (pol), HBV (pol) and B19 (ns) viral genomes and used to simultaneously detect individual viruses. In order to achieve optimal sensitivity and specificity for multiplex PCR, the thermo-cycling parameters, primer sequences, and concentration of each reaction components were optimized systematically. The sensitivity of the detection method ranged between 5 and 10 copies of viral genome with a mixture of multiple primer pairs. Furthermore, this highly sensitive test showed no cross-reactivity among the four viruses. Thus, the results obtained in this study provide evidence that the assay system is a good tool for supporting the diagnosis of viral infection and contamination.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.4
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• pp.293-300
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• 2005

연구목적: 단일 유전자 이상에 대한 착상전 유전진단을 성공적으로 시행하기 위해서는 효과적이고 신뢰도가 높은 PCR 방법의 확립이 중요하다. 본 연구에서는 alkaline lysis와 duplex nested PCR 방법을 단일 림프구와 할구의 유전자 분석에 적용하여 그 효용성을 확인하고자 하였다. 재료 및 방법: 단일 유전자의 이상이 확인된 Duchenne muscular dystrophy (DMD), ornithine transcarbamylase (OTC) 결핍증과 epidermolysis bullosa (EB) 가계의 대상자들에서 채취한 단일 림프구와 공여 받은 배아의 할구를 이용하여 각각 PCR, restriction fragment length polymorphism (RFLP)와 direct DNA sequencing 분석을 시행하였다. 이러한 분석에서 유전자 증폭률 (amplification rate)과 두개의 allele 중에서 하나의 allele이 증폭되지 않는 allele drop-out (ADO) 빈도에 대해 살펴보았다. 결 과: 단일 림프구와 할구를 이용한 PCR 방법의 유전자 증폭률은 DMD에서 91.1%와 81.8%, OTC 결핍증에서 96.0%와 78.1%, EB에서 91.3%와 90.0%를 각각 나타냈으며, ADO 빈도는 OTC 결핍증에서 13.3%, EB에서 16.8%로 관찰되었다. 결 론: 본 연구에서 적용한 alkaline lysis와 duplex nested PCR 방법은 단일 유전자에 대한 착상전 유전진단에 성공적으로 적용할 수 있는 방법으로 생각되며, ADO 빈도를 최소화할 수 있는 효율적인 방법의 개발에 대한 지속적인 연구가 필요하다.

• Korean Journal of Veterinary Research
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• v.42 no.4
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• pp.513-522
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• 2002

Antigenic ompC genes of S. gallinarum, S. pullorum and S. dublin were characterized among Salmonella spp. isolated from chickens and other animals to identify genetic variation. Salmonella ompC gene fragment (1,027 bp) was amplified by PCR and the amplicons were cloned for comparison of nucleotide sequences. The identity of the sequences between S. gallinarum and S. pullorum, S. gallinarum and S. dublin, S. pullorum and S. dublin was 99.8%, 97.6% and 97.8%, respectively. Also, we found that ompC has some diversity between S. gallinarum and S. pullorum, and other Salmonella spp. which may be useful to type the organisms. Similar to diagnosis in other organisms, the TaqMan PCR method can be applied to rapid and accurate diagnosis of salmonellosis in chickens and other animals. We designed PCR primers and TaqMan probe for flagellin gene (fliC) for detection of Salmonella spp. by TaqMan PCR. The TaqMan PCR method was 10,000 times more sensitive than conventional PCR.

• Genomics & Informatics
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• v.21 no.2
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• pp.24.1-24.7
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• 2023

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

• Journal of Oral Medicine and Pain
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• v.19 no.2
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• pp.205-219
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• 1994

Cigarette butts from 5 smokers were gathered and then, placed in room temperature for 1, 3, 5, 7, 15 days. The possible use of the cigarette butts for individual identification was evaluated in sex determination, amplification of D1S80 locus, polymorphisms of HLA-DQA1 gene from the extracted DNA. 1. DNA extraction was possible in cigarette butts weree left in room temperature for 15days, so it can be applicatable to individual identification by polymerase chain reaction(PCR). 2. Amplification of X-Y homologous amelogenin gene by PCR made it possible to identify the sex in saliva stains (cigarette butts). 3. Amplification of D1S80 locus can be acquired from adding the boving serum albumin and hot start PCR procedures from forensic samples such as saliva stains (cigarette butts), so the AMP-FLPs examining is possible. 4. Genotype could be determined simply and rapidly using Amplitype$TM$ HLA-DQ$\alpha$ forensic kit in examining the HLA-DQA1 gene. From the investigation, DNA extraction, sex determination, amplification of D1S80 locus, polymorphisms of HLA-DQA1 gene was successfully done even though the cigarette butts were left for 15 days at room temperature. Therefore cigarette butts are highly reliable and applicatable as molecular biologic samples for individual identification.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.555-559
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• 2013

Vibrio cholerae O1 and O139 are the major serotypes associated with illness, and some V. cholera non-O1 and non-O139 isolates produce cholera toxin. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the species-specific detection and quantitation of V. cholera using a primer pair based on an outer membrane lipoprotein lolB gene for the amplification of a 195 bp DNA fragment. The qPCR primer set for the accurate diagnosis of V. cholera was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.