• Journal of Microbiology and Biotechnology
• /
• v.17 no.10
• /
• pp.1616-1621
• /
• 2007

We have established a SYBR Green-based realtime PCR method using AnyDirect solution, which enhances PCR from whole blood, for direct amplification of the virA gene of Shigella flexneri and the invA gene of Salmonella typhimurium from human feces without prior DNA purification. When we compared the efficiency of conventional or realtime PCR amplification of the virA and invA genes from the supernatant of boiled feces supplemented with S. flexneri and S. typhimurium in the presence or absence of AnyDirect solution, amplification products were detected only in reactions to which AnyDirect solution had been added. The detection limit of real-time PCR was $1{\times}10^4\;CFU/g$ feces for S. flexneri and $2{\times}10^4\;CFU/g$ feces for S. typhimurium; this sensitivity level was comparable to other studies. Our real-time PCR assay with AnyDirect solution is simple, rapid, sensitive, and specific, and allows simultaneous detection of S. flexneri and S. typhimurium directly from fecal samples without prior DNA purification.

• Korean Journal of Environmental Agriculture
• /
• v.30 no.4
• /
• pp.367-376
• /
• 2011

BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

• Pediatric Gastroenterology, Hepatology & Nutrition
• /
• v.3 no.1
• /
• pp.41-46
• /
• 2000

Purpose: It is important to have the epidemiologic data of rotavirus serotypes for the application of polyvalent rotavirus vaccines. Epidemiological studies of rotavirus serotypes in Korea have been reported only in limited areas with small number of cases. Authors tried to investigate the distribution of rotavirus G serotypes in ChungJu area with RT-PCR. Method: Stool specimens were collected from 202 children with acute diarrheal symptoms, who admitted to or visited Kon-Kuk University Hospital in ChungJu from June 1998 to May 1999. Samples were screened for rotavirus with EIA method (TestPack Rotavirus, Abbott Laboratories) and rotavirus G Serotypes were determined by RT-PCR. Results: Rotavirus was positive in 46.6%. The incidence of G serotypes was as follows; G1 10%, G2 10%, G3 28%, G4 26%, and G9 20%. There were three cases of multiple serotypes; G1 with G9, G2 with G9, and G4 with G9. Serotype of G8 was not found. Conclusion: The proportion of G serotypes in ChungJu is much different from previous reports. Serotype of G9 was found which had not been reported in Korean children till now. Long term plans for the investigation of rotavirus serotypes must be needed in wide area.

• Journal of Life Science
• /
• v.13 no.5
• /
• pp.651-657
• /
• 2003

Randomly amplified polymorphic DNA (RAPD) analysis was used to study genetic relationships among C. polykrikoides, G. impudicum and G. catenatum, which possess similar morphological features. Four of 12 primers were selected and 59 amplification products ranged from 0.2 kb to 3.0 kb. The number of polymorphic products in C. polykrikoides, G. impudicum and G. catenatum was 16 (27.1%), 8 (13.5%), and 16 (27.1%), respectively, while 17 were monomorphic. Number of species-specific bounds was 26 (44.0%), 34 (57.6%), 26 (44.0%) in C. polykrikoides, G. impudicum and G. catenatum, respectively. The genetic similarity between C. polykrikoides and G. impudicum/G. catenatum was 0.83, whereas G. impudicum and G. catenatum was 0.78. Our results suggest that C. polykrikoides, G. impudicum and G. catenatum are extremely different on the basis of RAPD analysis, despite similarity based on their morphology. The RAPD technique appears to be efficient in detecting genetic variation in these dinoflagellates.

• Parasites, Hosts and Diseases
• /
• v.55 no.6
• /
• pp.607-611
• /
• 2017

Primary maternal infection with toxoplasmosis during pregnancy is frequently associated with transplacental transmission of the parasite to the fetus. This study was conducted to test the utility of PCR assay to detect recent infections with Toxoplasma in aborted women at various gestational ages who referred to Obstetrics and Gynecology Department of Alavi Hospital in Ardabil during 2014 and 2016. Two hundred women with a history of single or repeated abortion were investigated in this study. Blood samples were tested for specific anti-Toxoplasma IgM and IgG antibodies by ELISA. According to the results, 53.5% of the women under study were positive for anti-Toxoplasma antibodies: 4.0% of them had IgM, 43.0% had IgG, and 6.5% had both IgM and IgG. Subsequently, Nested-PCR analysis was used to detect T. gondii DNA in the placenta of subjects. In 10.5% of the women, the results were positive for 529 bp element of T. gondii. Among them, 5 (23.8%) cases were IgM positive, 1 (4.8%) case was IgG positive, and 11 (52.4%) were both IgM and IgG positive. In 4 (19.0%) patients, none of the antibodies were found to be positive. In total, 16 patients had positive results in both ELISA and PCR methods, and 174 cases had negative results for new infection. The findings of this study revealed that T. gondii might be one of the significant factors leading to abortion, and that the analysis of placenta can be important in order to achieve increased detection sensitivity.

• Journal of Food Hygiene and Safety
• /
• v.22 no.4
• /
• pp.248-256
• /
• 2007

In this study, the competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Salmonella spp. in pork meat. After comparing three DNA extraction methods, the modified guanidine thiocyanate-phenol-chloroform method was chosen for Salmonella DNA extraction in artificially inoculated pork meat. The previously reported 284-bp invA gene (Rahn et al. Mol. Cell. Probes 1992) was tested for specificity, and 57 Salmonella strains and 24 non-Salmonella strains were evaluated. All Salmonella strains tested were invA positive, and all non-Salmonella strains produced no false positive amplification products. The detection limit achieved was as low as 1,460 colony-forming units (cfu) per 0.1g of pork meat. For cPCR, the invA gene, which features a 82 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA, which has the same primer binding sites, was co-amplified with Salmonella chromosomal DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to the cfu from the most probable number (MPN) method. Finally, the whole procedure took only 5 hr.

• Genomics & Informatics
• /
• v.17 no.4
• /
• pp.42.1-42.6
• /
• 2019

Robust identification of genetic alterations is important for the diagnosis and subsequent treatment of tumors. Screening for genetic alterations using tumor tissue samples may lead to biased interpretations because of the heterogeneous nature of the tumor mass. Liquid biopsy has been suggested as an attractive tool for the non-invasive follow-up of cancer treatment outcomes. In this study, we aimed to verify whether the mutations identified in primary tumor tissue samples could be consistently detected in plasma cell-free DNA (cfDNA) by digital polymerase chain reaction (dPCR). We first examined the genetic alteration profiles of three colorectal cancer (CRC) tissue samples by targeted next-generation sequencing (NGS) and identified 11 non-silent amino acid changes across six cancer-related genes (APC, KRAS, TP53, TERT, ARIDIA, and BRCA1). All three samples had KRAS mutations (G12V, G12C, and G13D), which were well-known driver events. Therefore, we examined the KRAS mutations by dPCR. When we examined the three KRAS mutations by dPCR using tumor tissue samples, all of them were consistently detected and the variant allele frequencies (VAFs) of the mutations were almost identical between targeted NGS and dPCR. When we examined the KRAS mutations using the plasma cfDNA of the three CRC patients by dPCR, all three mutations were consistently identified. However, the VAFs were lower (range, 0.166% to 2.638%) than those obtained using the CRC tissue samples. In conclusion, we confirmed that the KRAS mutations identified from CRC tumor tissue samples were consistently detected in the plasma cfDNA of the three CRC patients by dPCR.

• Parasites, Hosts and Diseases
• /
• v.44 no.4 s.140
• /
• pp.343-353
• /
• 2006

Giardia intestinalis infections arise primarily from contaminated food or water Zoonotic transmission is possible, and at least 7 major assemblages including 2 assemblages recovered from humans have been identified. The determination of the genotype of G. intestinalis is useful not only for assessing the correlation of clinical symptoms and genotypes, but also for finding the infection route and its causative agent in epidemiological studies. In this study, methods to identify the genotypes more specifically than the known 2 genotypes recovered from humans have been developed using the intergenic spacer (IGS) region of rDNA. The IGS region contains varying sequences and is thus suitable for comparing isolates once they are classified as the same strain. Genomic DNA was extracted from cysts isolated from the feces of 5 Chinese, 2 Laotians and 2 Koreans infected with G. intestinalis and the trophozoites of WB, K1, and GS strains cultured in the laboratory, respectively. The rDNA containing the IGS region was amplified by PCR and cloned. The nucleotide sequence of the 3' end of IGS region was determined and examined by multiple alignment and phylogenetic analysis. Based on the nucleotide sequence of the IGS region, 13 G. intestinalis isolates were classified to assemblages A and B, and assemblage A was subdivided into A1 and A2. Then, the primers specific to each assemblage were designed, and PCR was peformed using those primers. It detected as little as 10 pg of DNA, and the PCR amplified products with the specific length to each assemblage (A1, 176bp; A2, 261 bp; B, 319 bp) were found. The PCR specific to 3 assemblages of G. intestinalis did not react with other bacteria or protozoans, and it did not react with G. intestinalis isolates obtained from dogs and rats. It was thus confirmed that by applying this PCR method amplifying the IGS region, the detection of G. intestinalis and its genotyping can be determined simultaneously.

• Korean Journal of Clinical Laboratory Science
• /
• v.40 no.2
• /
• pp.113-117
• /
• 2008

It designed a study to examine the efficiency of DNA and RNA extraction from archival formalin-fixed, paraffin-embedded tissues using an non-heating and heating method. Archival paraffin blocks of liver, kidney, colon were randomly selected. Each paraffin block was prepared in 20 microtubes. For each paraffin blocks were tested non-heating DNA extraction to 10 microtubes and heating protocol under pH 7.0 and $100^{\circ}C$ to 10 microtubes. Evaluation of the results of DNA extraction was carried out by measuring concentration by UV spectrophotometry and then PCR amplification. DNA extraction content that non-heating method was liver $5{\pm}0.7{\mu}g/mL$, kidney $2{\pm}0.3{\mu}g/mL$, colon $6{\pm}0.4{\mu}g/mL$ and heating method was liver $12{\pm}0.6{\mu}g/mL$, kidney $7{\pm}0.5{\mu}g/mL$, colon $10.{\pm}0.3{\mu}g/mL$. Successful RNA extraction was observed, by ${\beta}$-actin amplification, in 46.7% sections for samples treated by the heating method versus 30.0% using non-heating DNA extraction. The extracted nucleic acid showed better values for samples heated at $100^{\circ}C$. Therefore heating extraction of nucleic acid is reliable, quick and efficiency.

• Korean Journal of Veterinary Research
• /
• v.38 no.1
• /
• pp.173-181
• /
• 1998

Esherichia coli O157 : H7의 slt I, slt II, uid A 및 eaeA 4종 유전자를 동시에 검출하기 위한 multiplex PCR 기법을 확립하고 쇠고기중 직접 E coli O157 : H7 검출시험을 실시하였다. 4 set의 primers를 이용한 multiplex PCR 기법으로 31종의 장내세균에 대한 특이성을 조사한 결과 E coli O157 : H7 에서 1,087bp (eae A), 584bp (slt II), 348bp (slt I) 또는 252bp (uid A)크기의 DNA를 동시에 특이적으로 검출할 수 있었다. E coli O157 : H7 15주는 모두 uid A 및 eae A 유전자가 동시에 검출되었고, 다른 장내세균에서는 검출되지 않았다. slt I 또는 slt II 유전자를 가지고 있는 E coli 표준균주 24종을 이용하여 multiplex PCR 기법과 Vero cell cytotoxicity assay을 비교검사한 결과 베로톡신 산생능과 PCR법의 결과는 일치하였다. mutiplex PCR 기법의 쇠고기중 검출한계는 modified EC(mEC)에서 증균없이는 E coli O157 : H7균 $10^4cells/g$ 이상에서 검출이 가능하였으나 mEC에 1차 증균후 modified TSB 증균하였을 경우에는 10cells/g이하까지도 검출이 가능하였다. 개발된 multiplex PCR 기법을 쇠고기 40종에 직접 적용한 결과 E coli O157 : H7은 검출되지 않았으나 slt I 및 slt II유전자를 가지고 있는 E coli 4종이 검출되었으며, 이들의 혈청형은 O6, O112, O115 및 O139 였다. 이 연구에서 개발된 multiplex PCR은 쇠고기중 E coli O157 : H7을 신속하고 특이적으로 검출하는데 사용할 수 있을 것으로 사료된다.