• Research in Plant Disease
• /
• v.11 no.1
• /
• pp.48-55
• /
• 2005

Cylindrocarpon destructans is a soil-borne plant pathogenic fungus causing root rot on ginseng and trees. Rapid and exact detection of this pathogen was practiced on ginseng seedlings by nested PCR using speciesspecific primer set. The second round of PCR amplification by Dest 1 and Dest 4 primer set formed 400 bp of species-specific fragment of C. destructans from the product of first round of amplification by ITS 1 and ITS 4 primer set. In the PCR sensitivity test based on DNA density, nested PCR detected to the limit of one fg and it meant the nested PCR could detect up to a few spores of C. destructans. Also, nested PCR made it possible to detect the pathogen from ginseng seedlings infected by replantation on artificial infested soil. Our nested PCR results using species-specific primer set could be utilized for diagnosis of root rot disease in ginseng cultivation.

• Journal of Genetic Medicine
• /
• v.9 no.1
• /
• pp.11-16
• /
• 2012

Purpose: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. Materials and Methods: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. Results: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. Conclusion: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.

• Journal of Veterinary Clinics
• /
• v.22 no.4
• /
• pp.348-352
• /
• 2005

We described a rapid, sensitive and reproducible real-time PCR assay for detection and quantitation of canine parvovirus type 2 in the feces of dogs with parvovirus infection. The method was demonstrated to be highly specific and sensitive, allowing a precise canine parvovirus type-2 quantitation over range of eight orders of magnitude from $10^2\;to\;10^9$ copies of standard DNA. Then, fecal samples from parvovirus infected dogs were analyzed by conventional PCR and real-time PCR. Real-time PCR is more sensitive than conventional PCR, allowing to detect low viral titers of CPV-2 in infected dogs. By real-time PCR, a wide range of parvovirus particles was found in the samples from $1.45\times10^6\;to\;9.45\times10^8$ copies/0.01g of feces. However, when dogs are in infection of parvovirus, it is difficult to prove that the numbers of peripheral blood leukocytes are correlated with those of fecal shedding virus.

• Research in Plant Disease
• /
• v.21 no.3
• /
• pp.180-185
• /
• 2015

In the present work, a pair of primers specific to Tomato yellow leaf curl virus (TYLCV) was designed to allow specific amplification of DNA fragments from any TYLCV isolates using an extensive alignment of the complete genome sequences of TYLCV isolates deposited in the GenBank database. A pair of primers which allows the specific amplification of tomato ${\beta}$-tubulin gene was also analyzed as an internal PCR control. A duplex PCR method with the developed primer sets showed that TYLCV could be directly detected from the leaf crude sap of infected tomato plants. In addition, our developed duplex PCR method could determine PCR errors for TYLCV diagnosis, suggesting that this duplex PCR method with the primer sets is a good tool for specific and sensitive TYLCV diagnosis. The developed duplex PCR method was further verified from tomato samples collected from some farms in Korea, suggesting that this developed PCR method is a simple and reliable tool for rapid and large-scale TYLCV detections in tomato plants.

• Journal of Microbiology
• /
• v.38 no.2
• /
• pp.105-108
• /
• 2000

Oligonucleotide primers amplifying a 344 bp fragment on the integrin-like protein alpha-INT1p gene (${\alpha}$INT1) of Candida albicans were synthesized for screenign of C. albicans from clinicalsamples by the polymerase chain reaction (PCR). The PCR specifically amplified DNA from C. albicans and none from any other Candida, fungal, or human DNA in standard used here. The PCR assay showed that the primers (LH1 and LH2) were specific for 26 isolates of C. albicans from clinical smaples, whereas the positive fragment, 344 bp, was not amplified from 15 clinical isolates including 14 other medically important Candida species and an isolate of Saccharomyces cerevisiae. PCR was conducted on the urine samples of 20 patients and 4 samples were C. albicans positive. The detection limit of the PCR assay for C. albicans was shown to be approximately 10 cells/ml saline. The PCR system using 344 bp ${\alpha}$INT1 as a target is more specific and rapid than the conventional culture method, and the sensitive detection method is applicable to clinical diagnosis of C. albicans infections.

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

• Tuberculosis and Respiratory Diseases
• /
• v.39 no.6
• /
• pp.517-525
• /
• 1992

Background: Since its development by Saiki et al, polymerase chain reaction (PCR) has been very useful in various fields of molecular biology. PCR can be used for the detection of a very small amount of microbial agent, and is especially useful in those patients who are difficult to diagnose microbiologically or serologically. Mycobacterium tuberculosis is a very slowly growing organism and AFB staining frequently shows false negative results, and therefore PCR would be a very rapid, easy, and sensitive diagnostic method for the diagnosis of Mycobacterium tuberculosis. Method: To compare PCR with conventional methods in diagnosing Mycobacterium tuberculosis in sputum, we used sputa of patients who visited or were admitted to Seoul National University Hospital. The amplification targets were 383 base pair DNA, a part of 2520 base pair DNA encoding 65 kD Mycobacterium tuberculosis specific protein (the primers are TB-1, -2), and 123 base pair DNA, a part of IS6110 fragment, which multiple copies are known to exsist PCR one genome (the primers are Sal I-1, -2). We also requested AFB staing and culture to the lab of Seoul National University Hospital with the same sample and compared the results. Results: 1) Using TB-1, -2 primers, PCR was positive in 73.1% (19/26) of culture positive sputa, in 12.5% (1/8) of culture negative. but clinically diagnosed tuberculous sputa, and was negative in all sputa of patients who were clinically diagnosed as non-tuberculous etiology. 2) Using Sal I-I, -2 primers, PCR was positive in 94.1% (32/34) of culture positive sputa, in 23.1% (6/26) of culture negative, but clinically diagnosed tuberculous sputa, and was negative in 87.5% (14/16) of sputa from patients who were clinically diagnosed as non-tuberculous etiology. Conclusion: PCR could be a very rapid, sensitive and specific method for the diagnosis of Mycobacterium tuberculosis in sputa, and further studies should be followed for the development of easier method.

• Korean Journal of Microbiology
• /
• v.41 no.3
• /
• pp.168-176
• /
• 2005

Vibrio vulnificus, one of the marine bacterial pathogens causing septicemia, was detected using molecular methods, namely, PCR and/or Southern hybridization, and real-time PCR. Extracted and purified total DNAs by using commercial kits were used as templates for PCR. Multiplex-PCR was conducted by employing three sets of primers for the genes, hemolysin (vvhA), phosphomannomutase (pmm), and metalloprotease (vvpE), for V vulnificus virulence. The presence of DMSO ($5\%$) and BSA ($0.1\%$) in PCR reaction mixture improved a detection efficiency by higher PCR band intensities. TaqMan real-time PCR was carried out by using gene segment of vvhA as a target. Detection limit of PCR/Southern hybridization without enrichments was to be around $10^2\;cells\;g^{-1}$ of sample. However, those three methods using the enrichment at $35^{\circ}C$ in APW showed high sensitivity ($2\~10\;cells\;g^{-1}$ of sediments). Highly sensitive detection of V vulnificus by real-time PCR was achieved within $5\~6$ hr, whereas the detection by PCR/Southern hybridization required about 36 hr. Thus, it was evident that real-time PCR is the most rapid and efficient method for detecting V vulnificus in tidal flat sediments.

• Journal of Apiculture
• /
• v.32 no.1
• /
• pp.27-39
• /
• 2017

PCR-chip-based ultra-rapid multiplex PCRs for detection of six major infectious pathogens in honeybee were developed. The 6 kinds of major infectious pathogens in honeybee included Paenibacillus larvae causing American Foulbrood, Melissococcus plutonius causing European Foulbrood as bacteria, Ascosphaera apis (Chalkbrood), Aspergillus flavus (Stonebrood), Nosema apis and Nosema ceranae (Nosemosis) as fungi. The developed PCR-chip-based ultra-rapid multiplex PCR showed successful amplification for all six major pathogens in the presence of more than $10^3$ molecules. The time for confirming amplification (Threshold cycles; Ct-time) was about 7 minutes for two species, and about 9 minutes for four species. Total 40 cycles of PCR took 11 minutes 42 seconds and time for melting point analysis was 1 minute 15 seconds. Total time for whole PCR detection was estimated 12 minutes 57 seconds (40 cycles of PCR and melting point analysis). PCR-chip based ultra-rapid multiplex PCR using standard DNA substrates showed close to 100% accuracy and no false-amplification was found with honeybee genomic DNA. Ultra-rapid multiplex PCR is expected to be a fast and efficient pathogen detection method not only in the laboratory but also in the apiary field.

• Korean Journal of Veterinary Service
• /
• v.39 no.2
• /
• pp.107-116
• /
• 2016

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 $TCID_{50}$/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.