• 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.

• Journal of Applied Biological Chemistry
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• v.58 no.4
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• pp.383-387
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• 2015

A duplex polymerase chain reaction (PCR) detection method was developed to authenticate the use of cat and rabbit in food and to prevent unlawful distribution of illegally butchered meat in both domestic and imported food market. Species-specific primers were designed targeting mitochondrial cytochrome b gene. The sizes of PCR products were 191 bp for cat and 101 bp for rabbit, which were relatively small for better application of the detection method on processed foods. Specificities of primers were verified using 21 animal species including cat and rabbit. Limit of detection was examined by serial dilution of the sample DNA and confirmed as 0.005 ng for rabbit and 0.0005 ng for cat using Bioanalyzer. The developed duplex PCR method showed specificity and sensitivity in the identification of two target species.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.801-806
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• 2002

A multiplex competitive polymerase chain reaction (PCR) method was developed for the rapid identification and quantification of Leuconostoc mesnteroides and Lactobacillus plantarum populations which are the key microorganisms in kimchi fermentation. The strain-specific primers were designed to selectively amplify the target genes encoding 165 rRNA of L. plantarum and dextransucrase of L. mesenteroides. There was a linear relationship between the band intensity of PCR products and the number of colony forming units of each model organism. The PCR quantification method was compared with a traditional plate-counting method f3r the enumeration of the two lactic acid bacteria in a mixed suspension culture and also applied to a real food system, namely, watery kimchi. The population dynamics of the two model organisms in the mixed culture were reliably predictable by the competitive PCR analysis.

• Korean Journal of Veterinary Research
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• v.57 no.2
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• pp.79-88
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• 2017

The study of canine vector-borne diseases in the Philippines started in the 1970s but only gained interest in the past decade. Characterization of such diseases in the Philippines remains incomplete, thus, it is necessary to obtain additional information on the prevalence and diversity of canine tick-borne diseases in the country. In this study, blood samples were obtained at two veterinary clinics in Metro Manila, Philippines from 114 dogs suspected of having canine tick-borne pathogens. Polymerase chain reaction (PCR) was performed on whole blood DNA extracts followed by sequencing, and the following pathogens were detected: Hepatozoon (H.) canis (5.26%), Babesia (B.) vogeli (5.26%), Ehrlichia (E.) canis (4.39%), and Anaplasma platys (3.51%). Additionally, a set of multiplex PCR primers were developed to detect H. canis, Babesia spp. (B. canis and B. vogeli), and E. canis in canine blood. Multiplex and conventional single-reaction PCR results for the 114 dog blood samples were similar, except for one H. canis sample. Multiplex PCR is, therefore, a useful tool in screening infected dogs in veterinary clinics. This study's results, together with those of previous studies in the country, show that canine vector-borne pathogens are an emerging veterinary concern in the Philippines.

• Korean Journal of Agricultural Science
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• v.48 no.4
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• pp.1051-1065
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• 2021

Recent times have seen sustained increases in genetically modified (GM) crops not only for cultivation but also for the utility of food and feed worldwide. Domestically, commercial planting and the accidental or unintentional release of living modified (LM) crops into the environment are not approved. Many detection methods had been devised in an effort to realize effective management of the safety of agricultural genetic resources. In order to develop event-specific polymerase chain reaction (PCR) markers for LM crops, we analyzed the genetic information of LM crops. Genetic components introduced into crops are of key importance to provide a basis for the development of detection methods for LM crops. To this end, a total of 18 varieties from four major LM crop species (maize, canola, cotton, and soybeans) were subjected to an analysis. The genetic components included introduced genes, promoters, terminators and selection markers. Thus, if proper monitoring techniques and single or multiplex PCR strategies that rely on selection markers can be established, such an accomplishment can be regarded as a feasible solution for the safe management of staple crop resources.

• Korean Journal of Veterinary Research
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• v.43 no.2
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• pp.231-237
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• 2003

Listeria (L.) monocytogenes in samples could not be detected occasioally by faster growth of other Listeria spp. especially L. innocua. The aim of this study was to develop the differential media and multiplex polymerase chain reaction (PCR) assays for the rapid detection of L. monocytogenes. L. monocytogenes colonies were characterized by their ${\beta}$-hemolysis with fluorescence under 366 nm UV light on the Listeria hemolysis agar (LHA). L. innocua, a species commonly present in foods, did not produce ${\beta}$-hemolysis on LHA. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua. The multiplex PCR assays were developed to distinguish from L. monocytogenes and other Listeria spp. with two pairs of primers. The primers were designed in 16S rRNA and listeriolysin O gene for specific amplification of all members of the genus Listeria and L. monocytogenes, respectively. The multiplex PCR assays produced 560 and 938 bp products in L. monocytogenes; only 938 bp products in the genus Listeria. The multiplex PCR assays could detect as little as 50 pg of L monocytogenes DNA. These results indicated that the differential media and multiplex PCR assays might be useful diagnostic tools for the rapid detection of L. monocytogenes.

• Gut and Liver
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• v.12 no.6
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• pp.648-654
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• 2018

Background/Aims: Dual priming oligonucleotide-based multiplex polymerase chain reaction (DPO-based PCR) can detect the presence of clarithromycin resistance without culture. The aim of this study was to investigate the cost-effectiveness of DPO-based PCR for Helicobacter pylori eradication. Methods: From 2015 to 2016, medical records of patients who received H. pylori eradication therapy were analyzed. Patients were divided into two groups: tailored group patients who were treated based on DPO-based PCR and empirical group patients. Eradication rate and medical cost, including diagnostic tests, eradication regimens, and $^{13}C$-urea breath tests, were compared between the two groups. Cost for one successful eradication was calculated in each group. The expected cost of eradication for empirical treatment was investigated by varying the treatment duration and eradication rate. Results: A total of 527 patients were analyzed (tailored group 208, empirical group 319). The eradication success rate of the first-line therapy was higher in the tailored group compared to that in the empirical group (91.8% vs 72.1%, p<0.01). The total medical cost for each group was $114.8{\pm}14.1U.S.$ dollars (USD) and $85.8{\pm}24.4USD$, respectively (p<0.01). The total medical costs for each ultimately successful eradication in the tailored group and in the empirical group were 120.0 USD and 92.4 USD, respectively. The economic modeling expected cost of a successful eradication after a 7- or 14-day empirical treatment was 93.8 to 111.4 USD and 126.3 to 149.9 USD, respectively. Conclusions: Based on economic modeling, the cost for a successful eradication using DPO-based PCR would be similar or superior to the expected cost of a successful eradication with a 14-day empirical treatment when the first-line eradication rate is ${\leq}80%$.

• Biomedical Science Letters
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• v.5 no.1
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• pp.95-100
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• 1999

It is generally difficult, time-consuming, and expensive for the clinical laboratory to detect vancomycin resistant enterococci (VRE). The aim of this study was to develop and evaluate the multiplex polymerase chain reaction (PCR) assay system as a diagnostic tool for the rapid detection of VRE from clinical samples and/or for the identification of VRE from the bacterial strains isolated from clinical specimens. Specific primers, designed from the nucleotide sequences respectively encoding the vanA, vanB, vanC-1, vanC-2/3 genes in enterococci, were coupled in a multiplex PCR assay system. With this multiplex PCR assay system, we investigated the incidence rates and types of VRE isolated from clinical samples. A total of 75 strains of enterococci were isolated in 3 general hospitals in Korea. Of these isolates, 36 strains showed a pattern of high-level vancomycin resistance which associated with vanA gene, whereas 18 strains showed low-level vancomycin resistance associated with vanC-1 or vanC-2/3 gene. Thus, multiplex PCR assay method established in this study could be applied for the rapid detection of VRE.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.193-206
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• 2022

Astringent persimmon (Diospyros kaki Thunb.) is an important fruit crop in Korea; it possesses significant medicinal potential. However, knowledge regarding the pathogens affecting this crop, particularly, viruses and viroids, is limited. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput transcriptome sequencing (HTS) were used to investigate the viruses and viroids infecting astringent persimmons cultivated in Korea. A one-step multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of the pathogens was developed by designing species-specific primers and selecting the primer pairs via combination and detection limit testing. Seven of the sixteen cultivars tested were found to be infection-free. The RT-PCR and HTS analyses identified two viruses and one viroid in the infected samples (n = 51/100 samples collected from 16 cultivars). The incidence of single infections (n = 39/51) was higher than that of mixed infections (n = 12/51); the infection rate of the Persimmon cryptic virus was the highest (n = 31/39). Comparison of the monoplex and mRT-PCR results using randomly selected samples confirmed the efficiency of mRT-PCR for the identification of pathogens. Collectively, the present study provides useful resources for developing disease-free seedlings; further, the developed mRT-PCR method can be extended to investigate pathogens in other woody plants.

• Food Science and Preservation
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• v.16 no.6
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• pp.965-970
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• 2009

PCR technology has been widely used to detect and quantify microbial pathogens in foodstuffs, because the technique is rapid, sensitive, and selective. In this study, detection of contaminating pathogenic bacteria on shells of chicken eggs was performed using both a commercial multiplex polymerase chain reaction (PCR) kit and a viable count method employing a selective medium. The PCR kit was capable of detecting Campylobacter jejuni, Escherichia coli O157:H7, Staphylococcus aureus, Bacillus cereus, Vibrio parahaemolyticus, Listeria monocytogenes, Yersinia enterocolitica, Salmonella species, and Shigella species. Using the PCR method, five bacterial species were detected from 30 samples (33.3%) of 90 batches of eggs commercially available in a market. PCR products from B. cereus, S. aureus, L. monocytogenes, Y. enterocolitica, and E. coli O157:H7 were detected, and the numbers and frequencies of positive samples were 17 (18.8%), 12 (13.3%), 15 (16.6%), 16 (17.7%),and 4 (4.4%), respectively. None of any Salmonella species, C. jejuni, V. parahaemolyticus, or Shigella species was detected in this study. The results of PCR testing were confirmed using a typical viable count method employing a selective medium. We suggest that the multiplex polymerase chain reaction (mPCR) assay is a rapid and reliable method for detection of pathogenic bacteria contaminating eggs.