• Korean Journal of Veterinary Research
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• v.36 no.3
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• pp.607-614
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• 1996

Several methods for rapid and accurate detection of VTe-producing E coli were established. These methods contain beta-glucuronidase-secretion test, beta-haemolysis-production test in blood agar, verocytotoxicity test, and PCR. All of the VTe-producing strains made beta-haemolysis on 5% sheep blood agar. VTe-producing strains secreted beta-glucuronidase whereas 0157:H7 strains producing VTI or VTII did not secrete that enzyme. Verocytotoxicity test was established for rapid diagnosis. VTe detection was rapider in Vero cell suspension than Vero cell monolayer. In PCR, there was a positive result only in VTe-producing E coli, not in VTI or VTII-producing E coli. In this experiment, 165 strains of E coli were islated from feces or intestinal contents of post-weaning piglets showing nervous sign or diarrhea. And 20 strains of E coli that produced VTe were selected by verocytotoxicity test and PCR. According to these experiments, there was a direct correlation between verocytotoxicity test and PCR. And verocytotoxicity test is recommended as a routine diagnostic method and PCR does as a accurate diagnostic method to detect VTe-producing E coli.

• Parasites, Hosts and Diseases
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• v.49 no.3
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• pp.281-284
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• 2011

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.

• Food Science and Biotechnology
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• v.14 no.3
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• pp.387-391
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• 2005

Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in pork meat. Pork meat was artificially inoculated with L. monocytogenes and DNA was extracted using guanidine thiocyanate-phenol-chloroform and subjected to PCR amplification. Sixteen primer sets for L. monocytogenes hlyA gene were tested for sensitive detection and the DG69/DG74 primer set was selected. The detection limit achieved with this primer set was as low as 860 colony-forming units (cfu) per 0.1 g of pork meat. When the samples were cultured at $30^{\circ}C$ for 16 hr in Brain Heart Infusion (BHI) medium, even a single bacterium could be detected with this primer set by PCR. For cPCR, the hlyA gene, which features a 148 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA which has the same primer binding sites was co-amplified with L. monocytogenes total DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to cfu from the Most Probable Number (MPN) method. The whole procedure took only 5 hr.

• Parasites, Hosts and Diseases
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• v.60 no.4
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• pp.295-299
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• 2022

Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.

• Korean Journal of Veterinary Research
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• v.44 no.1
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• pp.99-103
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• 2004

The aim of the present study was to develop a sensitive and specific assay for the diagnosis of alcelaphine herpesvirus 1 (AlHV-1) which is a cause agent of malignant catarrhal fever in ruminants. A1HV-1 is a gamma herpesvirus, which is frequent latent, and it is often difficult to detect its antigens or specific nucleic acids because of its low genomic copies in the infected tissues. In this study, polymerase chain reaction (PCR)-dot blot hybridization (DBH) assay for detecting AlHV-1 DNA was developed and evaluated for its sensitivity and specificity as comparison with PCR and DBH alone. The developed PCR-DBH was more sensitive than PCR or DBH alone and also very specific. The results showed that the sensitivity of PCR-DBH were higher and stronger than those of PCR and DBH alone. This PCR-DBH assay can be applied efficiently to confirm the presence of AlHV-1 virus on clinical samples and to differentiate specifically between AlHV-1 infection and other viral infections.

• Korean Journal of Food Science and Technology
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• v.33 no.5
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• pp.521-524
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• 2001

A method using PCR was developed for the monitoring of glyphosate tolerant soybean (GTS) produced by the DNA recombination technique. We designed 3 pairs of specific oligonucleotide primers based on the gene sequences inserted in soybean and in lectin and ferritin genes as internal standards. Template DNAs were isolated from soybeans by the modified hexadecyl trimethyl ammonium bromide (CTAB)method and used for PCR with different primer sets. PCR, used with specific primer sets for GTS detection, showed the amplified DNA fragments with GTS template DNA but no product showed with non-GTS template. PCR amplified products were confirmed by DNA sequencing and were detected for up to 0.05% of GTS template DNA.

• International Journal of Oral Biology
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• v.36 no.1
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• pp.1-6
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• 2011

The purpose of this study was to develop species-specific real-time quantitative PCR (RT-qPCR) primers for use in the detection of Aggregatibacter actinomycetemcomitans. These primers were designed based on the nucleotide sequences of the RNA polymerase ${\beta}$-subunit gene (rpoB). We assessed the specificity of the primers against nine strains of A. actinomycetemcomitans, eight strains (three species) of the Haemophilus genus, and 40 strains of 40 other oral bacterial species. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC $33384^T$. Our data reveal that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 2 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these qRT-PCR primers are suitable for application in epidemiological studies.

• 연구논문집
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• s.33
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• pp.17-25
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• 2003

In this work, thermal design of a PCR chip for LOC is systematically conducted. From the numerical simulation of a PCR chip based on the finite volume method, how to control the average temperature of a PCR chip and the temperature difference between the denaturation zone and the annealing zone is presented. The average temperature is shown to be controlled by adjusting heat input and a cooler as well as a heater is shown to be necessary to obtain three individual temperature zones for polymerase chain reaction. To reduce the time required, a heat sink for the cooler is not included in the calculation domain for the PCR chip and heat sink design is conducted separately by using a compact modeling method, the porous medium approach.

• Journal of Life Science
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• v.10 no.2
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• pp.182-187
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• 2000

The optimized RAPD-PCR conditions, which can be utilized as a basic information for the analysis of the genetic characteristics were investigated with four onion varieties, named Changryungdaego, Yeoeuijuhwang, Yakwangju, and Dabonghwang using Operon primers, OPR01 (TGCGGGTCCT) and OPZ20 (ACTTTGGCGG). We tested several concentrations of DNA, primer, and MgCl2, annealing temperature, number of PCR cycle, and presence/absence of pre-heating time at the begining of PCR reation in the 25${mu}ell$ volume. The best RAPD profiles were obtained using 50ng of DNA, 5mM of primer, 1.5mM of MgCl2, 45$^{\circ}C$ of annealing temperature and an absence of pre-heating time. An establishment of the stable and reproducible RAPD-PCR conditions are expected to be useful for the subsequent RAPD-related investigation, such as genetic characterization of the onion strains, re-establishment of phylogenetic relationships and development of new varieties.

• Journal of Veterinary Clinics
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• v.15 no.1
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• pp.140-145
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• 1998

The present study was carried out for the rapid and accurate detection of Anaplasma marginale in cattle using Polymerase Chain Reaction. One pair of primer, BAP-2 and AL34S, were designed to amplify a 409 Up fragment of the A marginale membrane surface protein encoding beta($msp{\beta}l$) gene with a hilly sensitive and specific PCR. A marginale isolated from naturally infected calf in Chonbuk area were used to obtain target genomic DNA for PCR. This study showed that a 409 bp of $msp{\beta}l$ gene fragment could be detected as little as 15 fg of purified A marginale genomic DNA. The amplified fragment with PCR was checked for the identification of $msp{\beta}l$ gene by enzyme restriction and sequencing. Also, the target DNA extracted directly from blood were used in the PCR reactions without prior purification to shorten the detection time. The PCR in the present study was considered convenient and rapid method for the detection of A marginale in whole blood of infected cattle.