• Parasites, Hosts and Diseases
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• v.51 no.6
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• pp.651-656
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• 2013

Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were $84.5{\pm}0.07^{\circ}C$ and $85.7{\pm}0.07^{\circ}C$, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.

• Journal of Microbiology
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• v.40 no.4
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• pp.282-288
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• 2002

Since culture of Helicobacter pylori is relatively insensitive and cumbersome, molecular detection and typing of H. pylori isolates are gaining importance for strain differentiation. In the present study genomic DNA of 42 gastric biopsies and H. pylori isolates from corresponding patients were analyzed and compared by PCR-based RFLP assay. The 1,132-bp product representing an internal portion of ureC gene of H. pylori was amplified by PCR and digested with restriction enzymes HindⅢ, AiuⅠ and PvuⅠ. The HindⅢ, AluⅠ and PvuⅠ digestion produced 4, 7, and 2 distinguishable RFLP patterns respectively from 42-H. pylori isolates. By combining all three restriction enzyme digestions, 15 RFLP patterns were observed. However, when PCR products from 42 gastric biopsy specimens were digested by restriction enzymes HindⅢ, AluⅠ and PvuⅠ, we observed 5, 8 and 2 RFLP patterns, respectively. Patterns from 34 of 42 gastric biopsy specimens matched those of corresponding H. pylori isolates from respective patients. Patterns from the remaining eight biopsy specimens differed and appeared to represent infection with two H. pylori strains. The patterns of one strain from each of these biopsies was identical to that of the isolate from corresponding patients and the second pattern presumably represented the co-infecting strain. From the study, it appears that PCR-based RFLP analysis is a useful primary tool to detect and is distinguish H. pylori strains from gastric biopsy specimens and is superior to culture techniques in the diagnosis of infection with multiple strains of H. pylori.

• Korean Journal of Veterinary Research
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• v.42 no.4
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• pp.495-504
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• 2002

Streptococcus suis is an important swine pathogen in nearly all countries with an extensive pig industry. It is associated with meningitis, arthritis, endocarditis, septicaemia, bronchopneumonia and sudden death. Attempts to control the disease are still hampered the lack of effective vaccines and sensitive diagnostic tools. A PCR method which can be used for the detection of virulent strains of serotype 2, which is most prevalent serotype, and serotype 1 was developed. However, serotype 1, 2, 7 and 9 strains are frequently isolated from diseased pigs. In European countries, S suis serotype 2 is the most prevalent type isolated from diseased pigs, followed by serotype 9 and 1. In Japan, capsular serotype 2 was also the most prevalent serotype, followed by capsular serotype 7. Most of S suis isolated from diseased pigs belong to a limited number of capsular serotype, often those between 1 and 9. We investigated the distribution of S suis serotype 1, 2, 7 and 9 from 740 pig lungs at abattoir in Jeolla and Chungcheong by rapid multiplex PCR assay. Fifty of 740 lung samples, 6.8%, were S suis postitive and identified S suis were divided by 38% (19/50) in serotype 2, 2% (1/50) in serotype 7 and 4% (2/50) in serotype 9. The distribution of S suis serotype in Korea was similar to other countries. Moreover, the multiplex PCR assay may be an useful diagnostic tool for the detection of pigs carrying serotype 1, 2, 7, 1/2, 9 and 14 strains in epidemiological and transmission studies and facilitate control and eradication programs.

• The Plant Pathology Journal
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• v.35 no.6
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• pp.654-661
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• 2019

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea's soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R² = 0.996, slope = -3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.

• Fisheries and Aquatic Sciences
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• v.9 no.2
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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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.4
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• pp.212-215
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• 2002

Tyrosinase transcript In the blood Is known as the marker of malignant melanoma and it has been often determined by using reverse transcription-polymerase chain reaction (RT-PCA) . However, after the PCR process, the quantification of amplified CDMA by the gel electrophoresis is not reliable and time-consuming. for this reason, we tried to quantify the PCR product using a cuvette-type biosensor, where the oligonucleotide probe was immobilized on the cuvette surface and the single strand CDMA, the denatured PCH product, was then hybridized onto the immobilized probe to give a response signal. The response was Immediate and takes 15 min to obtain a stable signal. The biosensor was much more sensitive comparing to the gel electrophoresis method. The quantification of PCR product using a cuvette-type biosensor was feasible and rapid.

• Environmental Engineering Research
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• v.20 no.1
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• pp.105-109
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• 2015

Triclosan is a synthetic antimicrobial agent used in numerous industrial and personal care products. Triclosan collected in wastewater treatment plants can be biodegraded up to 80%. However, little is studied about the abundances of known triclosan-degrading bacteria in activated sludge systems. A previous study reported that Sphingopyxis strain KCY1 isolated from activate sludge can cometabolically degrade triclosan. Recently, a quantitative PCR (qPCR) assay specific to strain KCY1 has been developed. Thus, this study investigated the abundance of strain KCY1 in three different activated sludge wastewater treatments using a qPCR assay. Additionally, ammonia-oxidizing bacteria (AOB), known as triclosan-degraders, and amoA gene were quantified. Strain KCY1 were detected in activated sludge samples from three different wastewater treatment plants. The concentrations of strain KCY1 and AOB were on the order of $10^5-10^6$ gene copies/mL, while amoA gene concentration was on the order of $10^4$ gene copies/mL.

• Fisheries and Aquatic Sciences
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• v.17 no.3
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• pp.339-344
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• 2014

PCR amplification with universal primer is a useful tool for speciation of symbionts in marine eukaryote coupled with robust separation method such as denaturing high performance chromatography (DHPLC). To overcome the biased amplification, clamping PCR is recommended to suppress the amplification of host gene. In this study, we evaluated the efficiency of rare gene detection for two kinds of clamping probes which were successfully utilized for eukaryotic symbiont analysis: C3 linked nucleotide (C3) and peptide nucleic acid (PNA). PNA was 3-4 orders of magnitude higher than that of C3 tested in clamping efficiency and rare gene detection. This represented that PNA could be a more competent clamping probe for the enhancement of PCR amplification for rare symbiont genes.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1475-1481
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• 2009

Anthrax is a zoonotic disease caused by Bacillus anthracis, and well recognized as a potential agent for bioterrorism. B. anthracis can be identified by detecting the virulence factors genes located on two plasmids, pXO1 and pXO2. The aim of the present study was to determine the presence of virulence genes in 27 isolates of B. anthracis isolated from clinical and environmental samples. For this purpose, multiplex PCR and DNA probes were designed to detect protective antigen (pag), edema factor (cya), lethal factor (lef), and capsule (cap) genes. Our results indicated that all the isolates contained all the above virulence genes, suggesting that the isolates were virulent. To the best our knowledge, this is the first study about the determination of virulence marker genes in clinical and environmental isolates of B. anthracis using multiplex PCR and DNA probes in India. We suggest that the above methods can be useful in specific identification of virulent B. anthracis in clinical and environmental samples.

• Journal of Microbiology
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• v.33 no.3
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• pp.252-259
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• 1995

The PCR was employed to detect and characterize the bovine immunodeficiency-like virus (BIV), which is a newly recognized member of the I entivirinae of the retroviruses. Degenerate primers representing the conserved regions in the pol genes of the Lentivirinae, were used to detect proviral DNA obtained from the bovine embryonic spleen cell cultures infected with BIV. The PCR amplified DNA fragment was molecularly cloned and sequenced. The BIV DNA fragment contained a sequence identical to that reported by Garvey et al. (Garvey et al., 1990. Virology, 175, 391-409). With the degenerate primers, peripheral blood mononuclear cells (PBMCs) of sick cattle and cells cultured with BIV were tested to determine genetic variation of BIV pol conserved sequence. We found the sequence heterogeneity within cultures and most variations occurred at the third base of codons that would not lead to amino acid substitutions. Another change was GAG (Glu) to AAG (Lys) within the BIV isolates. Interestingly, the altered sequence is also found in other lentiviruses such as HIV-2, SIV mac, CAEV and EIAV.