• Parasites, Hosts and Diseases
• /
• v.34 no.3
• /
• pp.177-184
• /
• 1996

In situ hybridization was performed to detect rat heumocwstis ca4nii in the lung sections. Rats were immunosuppressed by weekly subcutaneous injection of 10 mg/kg methylprednisolone. On the 6th, 8th and 9th week of immunosuppression, the lungs were removed and fled in 10% neutral formalin. A 22 base oligonucleotide probe complementary to p. carinii 5S ribosomal RMh was commercially synhesized and its 3' terminal was labeled wiH biotin. In situ hybridization was performed utilizing manual capillary action technolog)r on the Microprobe system. p. cnrinii were detected along the luminal surface of alveolar pneumocytes, in exudate of alveolar cavities, and also in secretory material of bronchioles. In the 6th week group, positive reaction was observed focally in the peripheral region of the lung sections, but the reaction was observed diffusely in the 8th or 9th week groups. In comparison with Grocott's methenamine silver stain, in situ hybridization technique can detect the organism rapidly, and can detect trophic forms very well. Furthermore, no nonspecific reaction with other pathogenic fungi and protozoa was recognized. Therefore, in situ hybridization can be a good technique to detect p. carinii in the lungs of infected rats.

• Korean Journal of Clinical Laboratory Science
• /
• v.50 no.3
• /
• pp.261-266
• /
• 2018

To determine the clinical utility of an immunoblot test and RT-PCR-hybridization test, 160 samples from patients with a chronic HCV infection were analyzed by two tests. A total of 133 samples out of 150 positive samples were positive by RT-PCR-hybridization. The true positive rate of the immunoblot tests and the concordance rate of the two tests was 88.6% and 89.3%, respectively. Serotyping and genotyping were performed to evaluate the distribution of the HCV subtype in Korean isolates. HCV serotypes 1 and 2, and genotypes 1b and 2a were the most common sources of HCV infections in this group. In 49 cases studied with the serotypes and genotypes, serotypes 1 and 2 were 57.1% and 42.9%, respectively. Genotypes 1b, 1b/2b, 2a, 2a/2c, and 2b were 51.0%, 2.0%, 34.7%, 8.2%, and 4.1%, respectively. This study shows that immunoblot tests are more useful for screening HCV infections. The RT-PCR-hybridization test confirmed the HCV infection in patients with positive immunoblot test results. The serotype test is preferred over the genotype test for monitoring the progression or response to treatment. On the other hand, there were no significant differences in the response to an ${\alpha}$-interferon treatment of HCV infection with serotype type 1 or type 2 in Korea.

• Microbiology and Biotechnology Letters
• /
• v.20 no.4
• /
• pp.483-490
• /
• 1992

In order to understand the transfer behavior of a particular gene in water environments, kanamycin resistance ($Km^r$) gene was tracked by Southern hybridization with DNA probe in its conjugal transfer. A $Km^r$ natural bacterial isolate and genetically modified microorganisms (GMMs) constructed from the isolate were used as donor for conjugal transfer of the $Km^r$ gene. The transfer frequencies of the $Km^r$ gene from GMM strains were generally 10 to 100 times higher than those from the natural isolate. The conjugants obtained from GMM strains in LB broth had more plasmids newly appeared, and particularly the conjugants in A Wand FW waters revealed more rearrangement in their plasmids as a function of conjugation time. When plasmids of the conjugants obtained in LB broth were Southern hybridized with DNA probe of the $Km^r$ gene, the $Km^r$ plasmids in the conjugants were detected at the same position of the plasmids in donor cells, in spite of the fact that the plasmids were highly rearranged in conjugant cells. But the $Km^r$ plasmids in the donor of DKI and DKC601, and DKC600 were not identified in the conjugants obtained after 50 h conjugation in AW and after 30 h in AW, respectively. The size of the $Km^r$ plasmids showing hybridization signal were a little changed in the conjugants obtained in A Wand FW waters. Therefore, the method of Southern hybridization with DNA probe was proved to be very specific and useful for tracking of particular genes in water environments.

• BMB Reports
• /
• v.42 no.2
• /
• pp.81-85
• /
• 2009

Early and accurate detection of drug resistant Mycobacterium tuberculosis can improve both the treatment outcome and public health control of tuberculosis. A number of molecular-based techniques have been developed including ones using probe molecules that target drug resistance-related mutations. Although these techniques are highly specific and sensitive, mixed signals can be obtained when the drug resistant isolates are mixed with drug susceptible isolates. In order to overcome this problem, we developed a new drug susceptibility test (DST) for one of the most effective anti-tuberculosis drug, isoniazid. This technique employed a microplate hybridization assay that quantified signals from each probe molecule, and was evaluated using clinical isolates. The evaluation analysis clearly showed that the microplate hybridization assay was an accurate and rapid method that overcame the limitations of DST based on conventional molecular techniques.

• 제어로봇시스템학회:학술대회논문집
• /
• 2004.08a
• /
• pp.196-201
• /
• 2004

In biometric and biomedical applications, a special transporting mechanism must be designed for the ${\mu}$TAS (micro total analysis system) to move samples and reagents through the microchannels that connect the unit procedure components in the system. An important issue for this miniaturization and integration is microfluid management technique, i.e., microfluid transportation, metering, and mixing. In view of this, this study presents an optimal fuzzy sliding-mode control (OFSMC) design based on the 8051 microprocessor and implementation of a complete microfluidic manipulated system implementation of biochip system with a pneumatic pumping actuator, a feedback-signal photodiodes and flowmeter. The new microfluid management technique successfully improved the efficiency of molecular biology reaction by increasing the velocity of the target nucleic acid molecules, which increases the effective collision into the probe molecules as the target molecules flow back and forth. Therefore, this hybridization chip was able to increase hybridization signal 6-fold and reduce non-specific target-probe binding and background noises within 30 minutes, as compared to conventional hybridization methods, which may take from 4 hours to overnight. In addition, the new technique was also used in DNA extraction. When serum existed in the fluid, the extraction efficiency of immobilized beads with solution flowing back and forth was 88-fold higher than that of free-beads.

• Environmental Mutagens and Carcinogens
• /
• v.18 no.2
• /
• pp.109-115
• /
• 1998

Fluorscence in situ hybridization with chromosome-specific probe has been shown to be a valid and rapid method for detection of chromosome rearrangements induced by chemical and physical agents. This method is useful for quantifying structural aberrations, expecially for stable ones, such as translocation and insertion, which are difficult to detect with conventional method in human lymphocyte. In order to use the FISH method as a biodosimeter for monitoring human population exposed to various chemical and physical agent, baseline level of chromosome rearragement was established. Blood from forty four healthy adults were collected and analysed with whole chromosome-specific probes by human chromosome 1,2 and 4. The frequencies of stable translocation were 2.45 per 100 cell equivalent and those of insertion, color juction, acentric and dicentric were 0.32, 3.28, 0.23 and 0.27 per 100 cell equivalent respectively. The frequencies of chromosome rearragements increased with age in both sexes except for dicenrics. From above result, stable aberrations accumulate with age and it may reflect integrated lifetime exposure of adverse environment.

• Molecules and Cells
• /
• v.23 no.1
• /
• pp.115-121
• /
• 2007

Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.

• Korean Journal of Veterinary Research
• /
• v.37 no.4
• /
• pp.809-816
• /
• 1997

In this presented study, we established a method for diagnosis of porcine epidemic diarrhea(PED) by in situ hybridization(ISH), which made distinct progress in diagnostic pathology. We also carried out the retrospective diagnosis through ISH to assume the exact time of the first outbreak and incidence of PED in Korea. The outbreak of PED in Korea reported in 1992. However, since the end of 1980's, some researches of pig-industry have already suspected the outbreak of PED, not transmissible gastroenteritis(TGE). In this experiment, we performed the ISH using 80 formalin-fixed and paraffin-embedded tissues of porcine intestine which were requests for pathological diagnosis from 63 farms whose primary sign was diarrhea from 1984 to 1991. We prepared biotinylated cDNA probe(492base pairs) for ISH by nick translation method and carried out the ISH, using $Microprobe^{TM}$ capillary action system(Fisher $Biotech^R$). We detected PED virus in intestinal mucosa of 2 cases in 1992, 1 case in 1988, and 1 case in 1987. As a result, we assume that the outbreak of PED in Korea have already started since 1987.

• Korean Journal of Veterinary Research
• /
• v.43 no.3
• /
• pp.477-483
• /
• 2003

Molecular diagnostic techniques have been used to identify porcine epidemic diarrhea virus (PEDV), a causative agent of acute enteritis in swine, but they were difficult to be petformed and time-consuming. To detect PEDV in a rapid and easy way, we developed biotinylated cDNA probe for N gene encoding the nucleoproteins of PEDV. Formalin-fixed and paraffin-embedded tissues from 24 naturally infected pigs were used for the experiment. The ISH produced a positive reaction in all cases. When intestinal tissues were hybridized with PEDV probe, strong signals were seen in the villus enterocytes of the jejunum and ileum. Hybridization signals were also found in the duodenum from one pig and in colon from dnother. In conclusion, ISH with a biotinylated cDNA probe was provided to be a useful diagnostic method for detecting PEDV effectively in routinely processed tissue sections.

• BMB Reports
• /
• v.34 no.1
• /
• pp.59-65
• /
• 2001

In subtractive hybridization, target sequences in the tester are enriched by hybridizing with an excess amount of driver, followed by removing the tester hybridized with the driver. All of existing subtractive cloning methods are designed to remove the tester/driver hybrid. The removal of hybrid, however, is often unsatisfactory For various reasons. In this study we developed a subtractive enrichment protocol in which the tester/driver can be completely removed by selecting only the tester/tester after hybridization. In this protocol both the tester and driver DNAs are ligated with same linker DNAs and amplified by polymerase chain reaction (PCR). The tester DNA is then digested with two different enzymes and used in subsequent hybridization with an excess driver. After hybridization, the DNA is ligated with the adaptor that is only compatible with the tester/tester. Since only the tester/tester can have the new adaptor, no tester/driver can be amplified by PCR in this protocol. Unlike other methods, a 100% subtraction efficiency can be achieved even though the enzymatic treatments used in the enrichment procedure are incomplete. Furthermore, only the hybridized tester DNA can have the new adaptor and be amplified by PCR, resulting in 100% denaturation in effect. The efficacy of this novel method was verified with the model system in which a known amount of the target sequence is included.