• 한국가축번식학회지
• /
• 제17권1호
• /
• pp.43-48
• /
• 1993

These experiments were carried out to construct mouse testis cDNA library and to to seen H-Y Ag gene. Mouse testis was obtained from BALB/c inbreed mouse that was after-born 1 week. Isolation of mouse testis total RNA was carried out by guanidum/cesium choloride, poly(A+) mRNAs were purified by oligo d(T)-cellulose chromatography method. To investigate protein synthesis activity, in-vitro translation carried out by total RNA and poly(A+) mRNA. The products of in-vitro translation were identified in 12.5% PAGE. Single strand DNA and double strand DNA were synthesized from poly(A+) mRNA and purified using phenol/chloroform/isoamylalcohol. Synthesized cDNA was combined with cohesive Eco RI polylinker, its recombination efficiencies were identified by X-gal and IPTG. In the cDNA library, 1$\times$107 phagemids were screened with 32P labelled probe. Hybridization were carried on $65^{\circ}C$ for 16~20hours. And 1$\times$106 phagemids were screened with rabbit-anti-H-Y. In former, select 5 positive clones, and later, 1 positive clone. Its southern blot analysis showed various size of insert cDNA from 0.7kb to 3kb.

• Journal of Microbiology and Biotechnology
• /
• 제14권2호
• /
• pp.284-289
• /
• 2004

A PCR assay for the rapid detection of Vibrio vulnificus strains was developed using a virulence gene for elastase found in various Vibrio species. The DNA sequences in the elastase gene facilitated the identification of a species-specific probe for pathogenic V. vulnificus strains from both clinical and environmental sources. Using an elastase gene-based PCR reaction, a species-specific 507-bp PCR product was visualized by agarose gel electrophoresis. Three different DNA extraction methods were then compared to improve the simplicity and rapidity of detection. A PCR assay using the conventional DNA extraction or boiling method was able to detect as few as 25 V. vulnificus cells, making the detection limits at least 1-log-scale lower than that for the EDT A-treated DNA extraction method. In particular, the boiling method, which does not require purification of the chromosomal DNA, was very effective in terms of simple and rapid detection. Meanwhile, the detection limit in a mixed bacterial culture that included other bacteria, such as Escherichia coli or Bacillus subtilis, was two V. vulnificus cells, which was 1-log-scale lower than that for the control. Accordingly, when coupled with a new DNA extraction method, the elastase gene-based PCR can provide a rapid, specific, and sensitive method for identifying V. vulnificus in clinical and environmental samples.

• Parasites, Hosts and Diseases
• /
• 제56권5호
• /
• pp.419-427
• /
• 2018

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, $FAM^{TM}$, $HEX^{TM}$, $Cy5^{TM}$, and CAL Fluor $Red^{(R)}$ 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was $2{\times}10$ copies for C. parvum and for C. cayetanensis, while it was $2{\times}10^3$ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

• Journal of Radiation Protection and Research
• /
• 제24권1호
• /
• pp.45-53
• /
• 1999

각 염색체에 특이한 DNA probe를 이용하는 FISH기법은 방사선에 의해 유발된 상호전좌 및 삽입 등의 염색체의 구조적 변화를 측정하는 매우 효과적인 방법으로서 그 활용성이 증가되고 있다. 본연구는 방사선 피폭시 생물학적 선량측정법으로서 FISH기법을 활용하기 위하여 사람의 1, 2, 4번 염색체에 특이한 probe를 이용하여 고선량 단일 피폭시 유발된 각종 염색체 이상빈도를 관찰하고 이를 PAINT분류체계에 의해 분석하였다. 방사선 조사에 의한 염색체 이상빈도는 상호전좌(t)와 이동원염색체(dic)의 수가 선량 증가에 따라 같이 증가하는 것을 알 수 있으며 color junction의 수도 선량에 따라 증가하는 것을 알 수 있었다. 상호전좌의 빈도는 이동원 염색체의 빈도보다 상대적으로 높게 나타났다. 삽입(ins), 무동원염색체(ace), 및 환상염색체(r)의 수도 선량 증가에 따라 같이 증가하는 것을 알 수 있었다. 기존의 염색체재배열 분석방법과 비교해 볼 때 FISH기법은 다양한 형태의 염색체재해열을 보다 쉽게 관찰할 수 있게 하며 생물학적 선량제로서 중요한 역할을 할 것이라 기대된다.

• 한국수의병리학회지
• /
• 제7권1호
• /
• pp.1-4
• /
• 2003

Malignant catarrhal fever (MCF) is a systemic disease of ruminants caused by a gamma herpesvirus, ovine herpesvirus 2 (OvHV-2). Dot blot hybridization (DBH) protocols for detecting and differentiating this MCF virus were developed. OvHV-2 specific primer pairs, 556/555, were used for the amplification of target DNA. Then, the amplified DNA was labeled with incorporation of digoxigenin (DIG). The Dig-labeled probe was able to detect and differentiate specifically OvHV-2 DNA. This DBH technique can be applied to confirm the presence of MCF virus on clinical samples and to differentiate specifically between OvHV-2 infection and other viral infections.

• Fisheries and Aquatic Sciences
• /
• 제24권11호
• /
• pp.351-359
• /
• 2021

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD_95%) was determined to be 5.3 viral genome copies/µL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

• 대한수의학회지
• /
• 제36권4호
• /
• pp.845-858
• /
• 1996

The purpose of this study was to establish early diagnostic methods for the detection of Aujeszky's disease viral antigens and nucleic acid in nasal cells, and buffy coats from experimentally infected living pigs by a combination of immunocytochemistry, in situ hybridization with digoxigenin(DIG)-labled probe and electron microscopy. Forty days old piglets were inoculated intranasally with $10^{7.0}TCID_{50}$ of Aujeszky's disease virus (ADV, NYJ-1-87 strain). The viral antigens and nucleic acid of ADV were detected in nasal cells, and buffy coat for 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopical method. The results were compared with conventional methods such as a porcine Aujeszky's disease serodiagnostic(PAD) kit, neutralization test(NT) and virus isolation. 1. The viral antigens, nucleic acids and capsids of ADV were detected in nasal cells, buffy coats from 3 days to 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopy, respectively. 2. When viral antigens were detected by the immunocytochemical technique, a diffuse brown deposit was observed in the nucleus and cytoplasm of nasal cells, buffy coats and PK-15 cells under a microscope. 3. DIG-labeled DNA probe was prepared by amplification of conserved sequence of recombinant ADV-gp50 clone with polymerase chain reacction. When ADV-DNA was detected by ISH with DIG-labeled probe, purplish blue pigmentation were observed in the nuclei and cytoplasms of ADV-infected cells under a microscope. Positive signals were observed in nasal cells and in the buffy coat and PK-15 cells at the first day after inoculation. 4. Where ADV-capsids were detected by transmission electron microscopical method, aggregation of capsids was observed in the nuclei and cytoplasms of nasal cells, buffy coats and PK-15 cells. The results suggested that these methods were considered as the highly sensitive and reliable tools for rapid and confirmative diagnosis of Aujeszky's disease in living pigs.

• Journal of Microbiology and Biotechnology
• /
• 제23권5호
• /
• pp.674-680
• /
• 2013

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/${\mu}g$ DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

• 한국동물위생학회지
• /
• 제30권4호
• /
• pp.505-510
• /
• 2007

This study was conducted to detect ${\beta}$-lactam antibiotic-resistant genes in the 400 E coli isolates from porcine fecal samples in Korea by a DNA chip. The DNA chip contains the specific probe DNAs of the ${\beta}$-lactam antibiotic-resistant genes that had been labeled with a mixture of primer set designed to amplify specific genes (PSE, OXA, FOX, MEN, CMY, TEM, SHV, OXY and AmpC) using a multiplex polymerase chain reaction (PCR). Of 400 isolates 339 contained at least one ${\beta}$-lactamases gene. Resistance to ${\beta}$-lactamases was mediated mainly by AmpC (n = 339, 100%), and followed by TEM (n = 200, 59.0%), CMY (n = 101, 29.8%), PSE (n = 30, 8.9%) and both OXA and SHV genes (n = 20, 5.9%), while the FOX, MEN and OXY genes were not detected. The other sixty-one did not contain any ${\beta}$-lactamase genes even though they were resistant to antimicrobial drugs. In conclusion, the DNA chip system can be used as a rapid and reliable method for detecting of ${\beta}$-lactamases genes, which will help veterinarians select the antibiotics for monitoring and treating of animal diseases.

• 대한수의학회지
• /
• 제33권1호
• /
• pp.43-51
• /
• 1993

Mitochondrial DNA(mt DNA) of Mammalian is the circular one which the 16.5K base pairs and show the maternal inheritance. Evolutional speed of nucleotide sequence is very fast. So that polymorphic analysis of mt DNA provide the useful informations to investigate the genetic relations of interspecies. Authors trials were focussed to compare with the polymorphic differences of mitochondrial DNA between Jindo and Japanese mongrel dogs. DNA was extracted from bloods of 21 head of Jindo dogs and 20 head of Japanese dogs and isolated using 10 kinds of restriction endonucleases(Apa I, BamH I, Bgl II, EcoR I, EcoR V, Hinc II. Hind III, Pst I, Sty I, Xba I) and then separated by the agarose gel electrophoresis. After sourthern blotting hybridization was completed using the mtDNA of Japanese mongrel dogs as a probe. Autoradiography was used to compare the polymorphism of mtDNA both dogs. The results obtained were as follows; 1. mt DNA of Jindo dog showed polymorphism resulting cleavage with four kinds of restriction endonuclease, Apa I, EcoR V, Hinc II, Sty I. While in the Japanese mongrel dogs observed the polymorphism in the five kinds of restriction endonuclease supplemented with EcoR I. 2. Compared with both dogs the frequency differences of DNA polymorphism were recognized in the specific restriction endonuclease Apa I. Consequently in the restriction endonuclease Apa I both dogs classified with three types as A, B, C however in the Jindo dogs frequency of C type was 71.5 percent but in Japanese mongrel dogs observed 45 percent in the A type. 3. DNA polymorphism obtained from the use of five kinds of restriction endonuclease were classified with seven types. In Jindo dogs frequency was highest in the type 6 as 71.4 percent but in the Japanese mongrel dogs showed 35 percent in the type 5. 4. Genetic distances calculated by NEI method showed 0.0089 in Jindo dogs and was 0.0094 in the Japanese mongrel dogs.