• Journal of Life Science
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• v.16 no.5
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• pp.729-733
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• 2006

Cryptosporidium is a waterborne pathogenic parasite which causes diarrhea. Immunomagnetic separation-immunofluorescent assay (IMS-IFA) has been a widely adopted for Cryptosporidium detection as standard method. However, this method does not provide information about viability or infectivity of Cryptosporidium. Therefore, many researchers have studied viability or infectivity analyses of Cryptosporidium with various methods such as vital staining, in vitro excystation, RT-PCR, cell culture, and mouse infection assay. In this study, two direct RT-PCR methods, cell culture RT-PCR and cell culture IFA were compared for sensitivity and other characteristics. The results showed that direct RT-PCR method with HSP70 genes had the highest sensitivity with detection up to 1 viable cell of Cryptosporidium. The infectious Cryptosporidium were detected up to 10 to 25 cells by cell culture methods in combination with RT-PCR and IFA. The infectious Cryptosporidium were apt to be quantified by cell culture IFA.

• Research in Plant Disease
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• v.16 no.2
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• pp.121-124
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• 2010

Immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) was applied to the detection of Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus (KGMMV), and Zucchini green mottle mosaic virus (ZGMMV) on Cucurbitaceae crops. These seed-borne tobamoviruses were accurately detected from the infected leaves and seeds by IC-RT-PCR. This method was estimated to be about 100 times more sensitive than ELISA, and also it allowed the direct confirmation of ELISA results by using the captured antigens from a completed ELISA microwell. This convenient and reliable method could be used routinely for large-scale field surveys or seed tests of Cucurbitaceae crops.

• BMB Reports
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• v.40 no.6
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• pp.986-1001
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• 2007

Cold acclimation improves freezing tolerance in plants. In higher plants, many advances have been made toward identifying the signaling and regulatory pathways that direct the low-temperature stress response; however, similar insights have not yet been gained for simple nonvascular plants, such as bryophytes. To elucidate the pathways that regulate cold acclimation in bryophytes, we used two PCR-based differential screening techniques, cDNA amplified fragment length polymorphism (cDNA-AFLP) and suppression subtractive hybridization (SSH), to isolate 510 ESTs that are differentially expressed during cold acclimation in Physcomitrella patens. We used realtime RT-PCR to further analyze expression of 29 of these transcripts during cold acclimation. Our results show that cold acclimation in the bryophyte Physcomitrella patens is not only largely similar to higher plants but also displays distinct differences, suggests significant alteration during the evolution of land plants.

• Research in Plant Disease
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• v.8 no.2
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• pp.92-100
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• 2002

For the survey of viruses infected in peanut cultivated in Korea, peanut seeds and leaves showing viral symptoms were collected from their growing areas. Typical symptoms on virus infected peanut leaves including mosaic, mottle with necrosis, yellowing, stripe or vein banding and stunts were observed. Two viruses isolated from the naturally infected peanuts were identified as Bean common mosaic virus(BCMV-PSt) and Peanut mottle virus(PeMoV) by their host range, immunosorbent elcetron microscopy(ISEM), direct immuno staining assay(DISA), RT-PCR, and intracellural symptoms. Direct negative staining method by electron microscope showed filamentous particles of about 780 m in length as well as inclusion bodies. In ultrathin sections of BCMV-PSt and PeMoV infected tissues, cytoplasmic cylindrical inclusions as well as filamentous virus particles were observed in the cytoplasm of parenchyma cells. ISEM revealed filamentous particles strongly decorated with antiserums of BCMV-PSt and PeMoV Peanut seeds were stained with BCMV-PSt and PeMoV antisera indicating the possibility of seed transmission far these viruses. Seedlings germinated from peanut seeds which reacted with antiserums of BCMV-PSt by DISA showed mild mottle or stripe symptoms while mosaic and necrotic mottle symptoms were observed for PeMoV-positive seedlings. Filamentous particles were strongly decorated with each antiserum under ISEM observation. BCMV-PSt coat protein gene of about 1.2 Kbp was amplified by RT-PCR. Altogether these results indicate that BCMV-PSt is the most prevalent virus infecting peanut in Korea.

• Journal of Microbiology and Biotechnology
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• v.32 no.9
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• pp.1098-1102
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• 2022

The placenta is a captivating multifunctional organ of fetal origin and plays an essential role during pregnancy by intimately connecting mother and baby. This study explicates placental pathology and information about 25 placentas collected from the mothers infected with novel coronavirus (SARS-COV-2). So far, congenital transmission of SARS-CoV-2 seems to be remarkably uncommon in spite of many cases of COVID-19 during pregnancy. Out of the 25 placental tissue samples collected, none has shown gene expression of SARS-CoV-2 when confirmed by RT-PCR. At the same time, nasal and throat swab samples collected from newborns of SARS-CoV-2-positive mothers correspondingly tested negative by RT-PCR. The shielding properties of placental barriers against viral infections from mothers to newborns remains a mystery. Major histopathological findings have been recorded as choriodecidual tissue with necrosis, intramural fibrin deposition, chorionic villi with fibrosis, and calcification. Moreover, although recent findings are insufficient to prove direct placental transmission of COVID-19, the abundance of angiotensin-converting enzymes-2 (ACE-2) on the placental surface could potentially contribute to unpleasant outcomes during pregnancy as SARS-CoV-2 gains access to human cells via ACE-2. Finally, the significance of these findings is vague and needs further study.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1470-1474
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• 2009

This paper describes the development a of direct multiplex reverse transcription-nested polymerase chain reaction (PCR) method, devised for simultaneous detection and typing of influenza viruses. This method combines the direct reverse transcription reaction without RNA purification with the enhancement of sensitivity and specificity of nested PCR. The method successfully detected three major human influenza viruses: influenza virus A subtype 1 (H1N1) and subtype 3 (H3N2), and influenza B virus (B). The minimum number of virus particles (pfu/ml) necessary for detection in spiked saliva samples was 200 (H1N1), 140 (H3N2), and 4.5 (B). The method's sensitivity and simplicity will be convenient for use in clinical laboratories for the detection and subtyping of influenza and possibly other RNA viruses.

• Research in Plant Disease
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• v.8 no.4
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• pp.207-214
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• 2002

Tobacco rattle virus(TRV) was detected from Gladiolus hybridus, Crocus spp. and Narcissus spp. leaves show-ing notched or stripe on the leaf and malformation symptoms collected from Daegu and Kyungbuk province by electron microscopy (EM), immunosorbent electron microscopy (ISEM) and host range study. Direct negative staining method by EM showed rigid rod long particles 170~200$\times$22 nm and rigid rod short particles 40~114$\times$22 m. TRV-K isolated from G. hybridus propagated with Nicotiana tabacum. TRV coat protein(CP) gene was amplified using specific oligonucleotide primer by RT-PCR. Sequence analysis of amplified CP gene showed 99.5％ nucleotide similarity to TRV-ORY.

• Biomedical Science Letters
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• v.19 no.2
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• pp.90-97
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• 2013

The tuberculin skin test (TST) and interferon gamma (IFN-${\gamma}$) release assay (IGRA) have been widely used for diagnosis of latent tuberculosis infection (LTBI). In order to overcome limitations of current LTBI diagnostic methods, the development of a novel molecular assay which is able to measure the IFN-${\gamma}$ messenger RNA (mRNA) expression level after stimulation with Mycobacterium tuberculosis (MTB) specific antigen was recently developed. The ability of a molecular assay to detect MTB infection was similar to commercial IGRA however, the optimal incubation time for stimulating IFN-${\gamma}$ was not yet established. Therefore, in this study the direct comparisons of MTB Ag stimulation times (4 and 24 hrs) were performed for diagnosis of MTB infection. Data showed that the coincident rate between QFT-GIT IFN-${\gamma}$ ELISA and IFN-${\gamma}$ RT-PCR (4 hrs) was 88.35% and that of QFT-GIT and IFN-${\gamma}$ RT-PCR (24 hrs) was 70.85%. Based on a receiver operating characteristic (ROC) curve, the 4 hrs-MTB specific Ag stimulation time for IFN-${\gamma}$ RT-PCR had the significant P value, 95% CI value, and AUC (P < 0.0001, 95% CI=0.82 to 1.02, and AUC=0.9214) in comparison with 24 hrs-MTB specific Ag stimulation time (P = 0.009, 95% CI=0.06 to 0.94, and AUC=0.7711). These results show that 4-hr was the most optimal MTB Ag stimulation time for performing IFN-${\gamma}$ RT-PCR. Although semi-quantitative RT-PCR had a few analytical limitations, it might be useful as an alternative molecular diagnostic method for detecting MTB infection.

• BMB Reports
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• v.30 no.3
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• pp.234-236
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• 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.

• Korean Journal of Food Science and Technology
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• v.39 no.1
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• pp.71-76
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• 2007

Foodborne illness caused by Noroviruses (NVs) is increasing rapidly in Korea. This study developed an effective detection protocol for NVs found in contaminated oysters and lettuce through an investigation using the major steps of virus particle separation, concentration and RT-PCR. As a surrogate model for NVs, the cultivable feline calicivirus (FCV) that belongs to the same Caliciviridae family was used. Instead of using a time-consuming ultracentrifugation method, efficient methods based on solvent extraction and PEG precipitation procedure were applied. Direct homogenization of a 25g sample of whole oyster and lettuce in 175mL PBS provided the simplicity that would be needed in the actual field of food product examination. The overnight PEG precipitation step at $4^{\circ}C$ was reduced to 3 h by placing the reaction tube in ice and by adjusting the PEG concentrations. The application of the use of chloroform and 0.2 ${\mu}m$ syringe filtration together showed a better detection efficiency than the use of chloroform alone in removing PCR inhibitors for both oyster and lettuce samples. Also, dilution of the extracted RNA solution before PCR provided increased sensitivity. The improved detection protocol developed in this study could be efficiently applied to detect FCV and most likely NVs from oysters and lettuce.