• 한국가축번식학회지
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• 제23권1호
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• pp.13-18
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• 1999

정소내 성숙한 정자를 생산하는 전능성을 가진 정조세포는 체세포와 동일수준으로 외래 유전자를 삽입 가능한 것으로 알려져 있다. 그러나, 이들 세포가 반수체 이후의 단계로 분화한 경우에는 왜래 유전자를 삽입하기보다는 단순히 결합하는 능력이 있는 것으로 알려져 있다 . 따라서, 본 연구는 외래 유전자를 정소실질내 주입함으로써 형질전환 동물생산이 가능한지에 대하여 검토하기 위하여, 한쪽 정소를 거세한 한국 재래산 양을 사용하였다. Liposome /DNA 복합체를 1 : 2의 비율로 희석한 후 정소실실내에 주입하여 정자를 경유한 유전자 전이의 가능성을 확인하였다. 또한 동결보존한 정액을 인공수정하기 위하여 PGF$_2$$\alpha$(0.15mg/kg/BW)를 근육 주사함으로써 인위적으로 발정을 유도한 후, 인공수정을 이용하여 임신과 분만을 유도하였다. 이들 결과를 요약하면 다음과 같다. 1. PCR에 의하여, 유전자 도입 후 채취한 정액에서 외래유전자는 80일 이상 존재하였으며, 가장 높은 전이율은 40 일째 얻어졌다. 이들 결과는 정조세포에 외래 유전자가 성공적으로 삽입되었음을 제안하였다. 2. 23 두 (평균 96 % 의 발정 유기율)의 재래산양에게 인공수정을 실시한 결과 이들 중 4두가 임신되어 7두의 자양이 생산되었다. 3. 생산된 7두의 산자중 genome DNA를 추출하여 PCR 및 Southern blotting을 실시 한 결과 2두가 형질전환으로 확인되었다. 이상의 결과로서 정자를 매개로 한 정소실질내 외래 유전자의 주입법은 형질전환의 생산을 위한 매우 유용한 수단으로 사용 가능함을 제안하였다.

• Parasites, Hosts and Diseases
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• 제55권6호
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• pp.601-606
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• 2017

Cystoisospora is responsible for morbidity in immunocompromised patients. PCR is sensitive for diagnosing Cystoisospora; however, it needs reevaluation for differential molecular diagnosis of cystoisosporiasis. We aimed at evaluating melting curve analysis (MCA) after real-time PCR (qPCR) in diagnosis and genotyping of Cystoisospora as an alternative to conventional PCR. We included 293 diarrheic stool samples of patients attending the Department of Clinical Oncology and Nuclear Medicine of Cairo University Hospitals, Egypt. Samples were subjected to microscopy, nested PCR (nPCR), and qPCR targeting the internal transcribed spacer 2 region (ITS2) of the ribosomal RNA (r RNA) gene followed by melting temperatures ($T_ms$) analysis and comparing the results to PCR-RFLP banding patterns. Using microscopy and ITS2-nPCR, 3.1% and 5.8% of cases were Cystoisospora positive, respectively, while 10.9% were positive using qPCR. Genotyping of Cystoisospora by qPCR-MCA revealed 2 genotypes. These genotypes matched with 2 distinct melting peaks with specified $T_ms$ at $85.8^{\circ}C$ and $88.6^{\circ}C$, which indicated genetic variation among Cystoisospora isolates in Egypt. Genotype II proved to be more prevalent (65.6%). HIV-related Kaposi sarcoma and leukemic patients harbored both genotypes with a tendency to genotype II. Genotype I was more prevalent in lymphomas and mammary gland tumors while colorectal and hepatocellular tumors harbored genotype II suggesting that this genotype might be responsible for the development of cystoisosporiasis in immunocompromised patients. Direct reliable identification and differentiation of Cystoisospora species could be established using $qPCR-T_ms$ analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.

• 대한수의학회지
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• 제36권1호
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• pp.119-129
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• 1996

The present study was conducted to rapidly detect Listeria monocytogenes in raw milk. Specificity and sensitivity of polymerase chain reaction(PCR) technique, and direct PCR were examinded in raw milk, also were compared the calssical culture methods with PCR technique. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L nomocytogenes. In the PCR specificity tests, each of the 10 strains of L monocytogenes tested gave a single 70-bp band. But the other six Listera spp tested gave negative results. Results of the sensitivity tests showed that as few as 2 CFU of L monocytogenes in pure cultures could be detected with 16S rRNA-based primers, L-1 and L-2. In different PCR cycles, a PCR product was detected with $10^3$ cells of L monocytogenes from 25 cycles to 50 cycles and the concentration of PCR products was cycle-dependent. Raw milk samopes added L monocytogenes cells gave negative results. However, these samplers gave a single 70-bp band by pretreatment of pronase, and PCR products were detected with $10^1$ cells of L monocytogenes. To detemine the most sensitive culture protocol to use in conjunction with the PCR assay, raw milk samples were inoculated with L monocytogenes at concentrations ranging from 1 to $5.7{\times}10^4CFU/ml$. PCR assays from Listeria enrichment broth(LEB) containing raw milk samples added L monocytogene EGD could dtect 10 cells in pronase-pretreated samples without incubation, and 1 cell of L monocytogenes in both 12 hr and 24 hr incubation, respectively. Isolation raw of PCR assays was similar to that of classical culture methods, but required time for detection of L monocytogenes could remarkably be reduced compare to culture methods.

• 한국식품과학회지
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• 제31권6호
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• pp.1628-1634
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• 1999

Listeria monocytogenes의 식품 속에서 신속하고 특이적인 검출을 위하여, listeriolysin O gene에 의한 primer, LM 1과 LM 2를 선택하여 PCR을 수행하였다. L. monocytogenes의 DNA 추출이나 cell lysis 없이 intact whole cell을 직접 이용하여 PCR을 하였으며 $10^{2-6}$ CFU 수준의 균체 배양액으로부터 L. monocytogenes에 특이적인 702 bp의 PCR 증폭 산물을 확인하였다. 우유, 닭고기, 김치 등의 식품에 L. monocytogenes를 접종하여 증균배양 전후 균의 PCR에 대한 감도와 생균수를 비교한 결과, 실험된 식품 속에서의 L. monocytogenes의 검출 감도는 순수 배양액에서의 경우에 비해 약 1/10로 둔화되었으나 역시 특이적인 검출이 가능하였다. Primer LM 1과 2를 이용한 본 실험조건에서의 L. monocytogenes의 PCR에 의한 검출은 약 4시간으로 확인이 가능하였으며, 기존의 배양 방법에 비해, 특이성이나 검출 속도 면에서 식품위생 실무에 적용하기 위한 높은 잠재력을 보여 주었다.

• Fisheries and Aquatic Sciences
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• 제9권2호
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• pp.70-74
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• 2006

DNA sequence-based typing is considered a robust tool for the discrimination of dinoflagellate species because of the availability of extensive rDNA sequences. Here, we present a rapid, cost-effective DNA-sequencing technique for various PCR products. This sequencing strategy relies on 'nested' or 'tailed' primer labeled with near-infrared dye, and uses a minimal volume of unpurified PCR product (ca. $5{\mu}L$) as the DNA template for sequencing reactions. Reliable and accurate base identification was obtained for several hundred PCR fragments of rRNA genes. This quick, inexpensive technique is widely applicable to sequence-based typing in clinical applications, as well as to large-scale DNA sequencing of the same genomic regions from related species for studies of molecular evolution.

• Tuberculosis and Respiratory Diseases
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• 제39권1호
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• pp.1-6
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• 1992

The diagnosis of tuberculosis is usually established using staining and culturing techniques. Fluorescent stains have improved the sensitivity of direct microscopy. Improved culture media coupled with radiometric means of detecting early mycobacterial growth have shortened the time needed for cultural diagnosis. Rapid immunodiagnostic techniques based on the detection of mycobacterial antigen or of antibodies to theses antigens have not, however, come into widespread clinical use. The DNA or RNA hybridization tests with labeled specific probes which have been described so far are not sensitive enough to be used for clinical speicimens without prior culturing. The advent of the polymerase chain reaction (PCR) has opened new possibilities for diagnosis of microbial infections. This technique has already been applied to a number of microorganisms. In the field of mycobacteria the PCR has been used to identify and to detect DNAs extracted from various mycobacteria. However, despite the extraordinary enthusiasm surrounding this technique and the considerable investiment, PCR has not emerged from the developmental "trenches" in the passed several years. It may be a considerable lenth of time before clinical microbiology laboratories become PCR playgrounds because many details remain to be worked out.

• Journal of Microbiology
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• 제34권3호
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• pp.236-240
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• 1996

Rifampin is the most powerful drug for treating leprosy and tuberculosis today. It inhibits initiation and elongation of RNA transcription by binding to $\beta$-subunit of RNA polymerase, leading to kill mycobacteria. We isolated one variant strain of Mycobacterium leprae from 24 Korean leprosy patients who are less susceptible to rifampin or have suffered from relapse by polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) of the rpoB gene. Direct sequencing of the rpoB region of M. leprae variant revealed missense mutations which altered the amino acids sequenceof RpoB to Ser-464, Arg-465, Arg-467 and Ala-468. This is the first finding on rpoB gene mutation of M. leprae from Korean patients ; moreover the mutant type was found to be different from the previously reported cases in other countries.

• 산업기술연구
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• 제28권A호
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• pp.141-147
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• 2008

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

• 대한수의학회지
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• 제41권4호
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• pp.529-533
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• 2001

칠면조에서 분리된 avian pneumovirus(APV)의 포유동물 전파 여부를 확인하기 위해 Balb/c 쥐에 감염을 시도하였다. APV롤 3주, 5주, 7주령의 실험동물에 비강내 감염시킨 후 감염동물과 동거동물의 임상증상 및 조직내 바이러스 유전자 존재 여부등을 확인하였다. 감염 동물 및 동거 동물에서 임상증상은 나타나지 않았으나 감염 6일후에 혈액, 폐, 기도, 구강 및 직장 시료에서 RT-PCR 방법으로 APV의 유전자를 검출할 수 있었다. 14일 후에는 혈액에서는 유전자가 검출되지 않았으나 혈청내 항체를 확인할 수 있었다. 동거동물에서는 폐와 직장시료에서 PCR로 APV를 검출할 수 있었다. 이와 같은 결과로 칠면조 유래 APV는 실험쥐에 감염되어 직접 접촉에 의한 전파도 가능하다는 것을 알수 있었다.

• Journal of Microbiology and Biotechnology
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• 제9권2호
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• pp.184-190
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• 1999

The use of the polymerase chain reaction (PCR) method was described using two sets of primers based on the ceuN gene (JEJ 1 and JEJ 2) which encodes a protein involved in siderophore transport and 16S rRNA gene (pA and pB) for the sensitive and specific detection of enteropathogen Campylobacter jejuni. Six oligonucleotides were utilized in an amplification experiment and PCR products of predicted sizes were generated from whole cells and boiled cell lysates at the same intensity. Two sets of the primer pairs, JEJ and pAB, were specific enough for all C. jejuni strains tested for the direct use of whole cells without DNA extraction or lysis steps. In the PCR using the pAB primer pair, the detection limit, as determined by the ethidium bromide staining of the amplification products on agarose gels, was at the level of $10^1$ bacteria cells or less in both the pure culture and artificially inoculated milk and chicken enrichment samples, whereas the detection limit with the JEJ primer pair was relatively low, i.e. $10^3$ cells or more in the same PCR samples. The PCR method using either a primer JEJ or pAB was both repeatable and specific for the detection of C. jejuni in food. This method is simply completed within 4 h.