• Parasites, Hosts and Diseases
• /
• 제53권2호
• /
• pp.147-154
• /
• 2015

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (${\approx}375bp$) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ${\approx}550bp$ of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ${\approx}2$ oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

• BMB Reports
• /
• 제30권3호
• /
• pp.234-236
• /
• 1997

Chronic hepatitis C virus (HCV) infection is associated with the rapid development of cirrhosis and hepatocellular carcinoma. It has been reported that the amount of HCV RNA may be correlated with the progression of hepatitis and may be a prognostic marker for treatment of HCV patients. The direct detection of HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR) is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. In the present study, we developed the method for HCV quantitation using competitive reverse transcription (CRT)-PCR using the deleted HCV standard. The serially diluted standard was added in titrated amounts to the target HCV RNA. The mixture was then reverse transcribed and amplified in the same reaction tube. The methods were evaluated using over 110 HCV-PCR positive samples in Koreans. About 59% of the samples were judged to contain $10^{5}-10^{6}$ copies of HCV RNA in 1 ml of serum.

• BMB Reports
• /
• 제37권1호
• /
• pp.1-10
• /
• 2004

DNA and RNA quantifications are widely used in biological and biomedical research. In the last ten years, many technologies have been developed to enable automated and high-throughput analyses. In this review, we first give a brief overview of how DNA and RNA quantifications are carried out. Then, five technologies (microarrays, SAGE, differential display, real time PCR and real competitive PCR) are introduced, with an emphasis on how these technologies can be applied and what their limitations are. The technologies are also evaluated in terms of a few key aspects of nucleic acids quantification such as accuracy, sensitivity, specificity, cost and throughput.

• 한국식품위생안전성학회지
• /
• 제34권3호
• /
• pp.277-282
• /
• 2019

본 연구에서는 한국 전통 식품에서 Salmonella Typhimurium와 Listeria monocytogenes의 검출에 대하여 LAMP에 기반한 3M Molecular Detection Assay 2 (3M MDA 2)와 식품공전에 등재된 분리배지, real-time PCR의 검출 성능을 비교하고자 하였다. 육회와 육사시미에 $10^0-10^4CFU/25g$의 수준으로 S. Typhimurium와 L. monocytogenes을 각각 접종하였다. Citrobacter freundii와 Listeria innocua는 S. Typhimurium와 L. monocytogene의 검출에 영향을 주는 균으로 사용하였다. S. Typhimurium와 L. monocytogenes만 $10^0-10^4CFU/25g$ 수준으로 접종한 모든 시료에서는 분리배지, real-time PCR과 LAMP에서 양성으로 검출되었다. C. freundii와 L. innocua를 같이 접종한 경우에서 부분적으로 양성이 나타났다. 육회와 육사시미에 대하여 real-time PCR 보다 3M MDA 2가 더 검출효율이 높음을 알 수 있었다. 분리배지가 가장 검출효율이 높았지만 3M MDA 2와 큰 차이가 없었다. 배지를 사용하는 방법은 최소 일주일의 시간이 소요되고 PCR의 경우 inhibitor의 영향을 많이 받아 정확한 검출이 어려운 점이 있다. 그러나 LAMP에 기반한 3M MDA 2는 enrichment 후 다음 날 간단한 protocol을 통해 25분 이내로 샘플의 양성 반응을 확인할 수 있어 식중독균에 대해 신속하고 정확한 검출이 가능한 것으로 판단되었다.

• 대한수의학회지
• /
• 제44권2호
• /
• pp.163-169
• /
• 2004

To elucidate the molecular mechanisms of autoimmune inflammation in the central nervous system, we examined the expression and localization of STAT1, STAT3, STAT4 and STAT6 molecules during experimental autoimmune encephalomyelitis (EAE) by competitive PCR. In the present study, we quantitated IL-4 and IL-12 p40 mRNA by competitive PCR in the CNS during EAE. IL-4 mRNA was found at early and peak stages. On the other hand, the IL-12 p40 mRNA level reached maximal levels at the peak stage and still found at the recovery stage of the disease. We examined the kinetics of STAT mRNA in the CNS during EAE and demonstrated that STAT1 and STAT4 mRNA reached a maximal level at the peak stage of EAE, whereas STAT3 mRNA level increased gradually to the recovery stage. STAT6 mRNA increased rapidly at the early stage followed by gradual decrease till the recovery stage. Taken together, these findings suggest that STAT4 which was probably activated by IL-12 plays a pro-inflammatory role and that STAT3 which was activated throughout the disease course seems to serve as a transducer of anti-inflammatory signals.

• 한국발생생물학회지:발생과생식
• /
• 제5권2호
• /
• pp.131-136
• /
• 2001

초파리 발생과정에서 세포사멸에 관여하는 유전자인 reaper(rpr), grim, dcp-1, diap1, diap1, diap2의 발현양상을 경제적 RT-PCR 방법으로 분석하였다. 세포사멸 유도 유전자인 rpr, grim의 발현양상은 발생단계에 따른 ecdysone titer 변화 양상과 매우 유사하였다. Effector caspase인 dcp-1 전사체는 초기 배와 암컷 성체에서 높은 발현을 보였다. 반면에 세포사멸 억제인자인 diap1과 diap2 전사체는 세포사멸 유도 인자인 rpr과 girm 전사체와 서로 상반적인 양상으로 발현되었다. 또한, 유주 3령 유충의 발생단계 별로 침샘조직과 성체원기조직에서 rpr, diap2, dcp-1의 전사체의 양적 변동을 분석하였다. rpr, diap2의 전사체양은 두 조직에서 서로 상반적으로 변화하였다. 이 결과는 정상 발생과정에서 세포죽음 관련유전자들의 발현이 ecdysone 신호에 의해 조절됨을 암시해 주었다.

• 한국미생물학회:학술대회논문집
• /
• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
• /
• pp.210.2-210
• /
• 2005

• 한국발생생물학회지:발생과생식
• /
• 제4권1호
• /
• pp.101-108
• /
• 2000

자궁내막증은 흔한 부인과적 질병이며 여성 불임의 한 원인이 되나 그 발생 원인에 대하여서는 아직 논란의 여지가 많다. 최근 월경혈의 역류에 의하여 자궁내막증이 생긴다는 가설이 가장 유력한데 자궁내막증 환자가 정상여성에서보다 역류되는 월경혈의 양이 많거나 침습성이 강한 것이 자궁내막증의 발생원인이 될 수 있다는 이론들이 소개되었다. Pleitrophin (PTN)이나 midkine(MK)은 성장 및 분화에 관여하는 인자로서 여러 종류의 악성 종양에서 그 발현이 보고되어있으며 종양화 (carcinogenensis), 맥관형성 (angiogenesis), plasminogen activator의 활성화 증가 등에 관여한다고 보고된 바 있다. 이에 자궁내막증 환자의 자궁 내막과 대조군의 자궁내막에서 PTN과 MK mRNA의 발현의 차이를 quantitative competitive RT PCR로 비교하였다. 그 결과 자궁내막증 환자의 황체기 자궁내막에서 대조군의 자궁내막에 비하여 PTN과 MK의 발현이 높게 나타났다. 이러한 PTN과 MK의 발현의 증가로 자궁내막증 환자의 자궁내막이 복강 내에서 더욱 쉽게 맥관형성을 하고 성장이 촉진되어 자궁내막증이 발생될 것으로 생각되어 PTN과 MK가 자궁내막증의 초기 발생과정에 관여할 가능성이 있다.

• The Plant Pathology Journal
• /
• 제17권5호
• /
• pp.295-299
• /
• 2001

The relative density of phytoplasmas in witches'broom (WB)-infected jujube trees was investigated using compatitive polymerase chain reaction (PCR). During dormant and defoliating seasons, the densities of phytoplasmas were about the same in roots and twigs. In early growing season, the density showed the highest rates in roots, then in twigs and in petioles. however, the density was highest in petioles and the lowest in roots during actively growing season. Throughout the year, root samples did not show any serious fluctuation compared with that of t2wigs and petioles. Density was lowest during actively growing season in root samples. In contrast, petiole sample densities varied to a great extent depending on the season, very high during actively growing season, but very low during the early growing season, In twig samples, the densities were very high and almost the same in both defoliating and dormant seasons. Among the parts of the trees, phytoplsma density was the most stable in root samples throughout the year. The highest densities of phytoplasmas were about the same in all tree parts. These results suggest that the phytoplasmas may overwinter not only in roots but also in twigs, and that multiplication rate of phytoplsma becomes very high right after the early growing season.

• Parasites, Hosts and Diseases
• /
• 제38권2호
• /
• pp.107-110
• /
• 2000

We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g. HSP70-NH2-terminal region, or rT.g. HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or rT.g.HSP70-carboxy-terminal region increased the number of T. gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NHa-terminal region did not. These results suggest that T.g. HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya.