• Parasites, Hosts and Diseases
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• v.53 no.1
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• pp.77-83
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• 2015

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.

• Research in Plant Disease
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• v.23 no.1
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• pp.65-68
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• 2017

Barley yellow mosaic virus (BaYMV), which is transmitted by the root-inhabiting protist Polymyxa graminis, causes a soil-borne disease. In this study, we detected BaYMV from soil using two-step nested polymerase chain reaction (PCR). Specific primers based on a coat protein region of BaYMV segment RNA1 were used in the first round of amplification. Based on the sequenced amplicon, an inner primer was designed for the second round of amplification. A PCR product of 372 bp exhibited 98%-100% nucleotide sequence identity with the coat protein region of BaYMV segment RNA1. In this study, we propose an easy method for the detection of BaYMV from soil, may considerably assist in accurate fungus-transmitted virus diagnosis and subsequent disease forecasting. This is the first report on the detection of BaYMV from soil.

• Journal of Veterinary Clinics
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• v.14 no.2
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• pp.177-184
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• 1997

Canine parvovirus(CPV) is a very highly contagious virus causing hemorrhagic enteritis and myocarditis mainly in young dogs. The diseases were first recognized in 1978, and then spread throughout the world by 1980. The main source of the infection seems to be the feces of infected dogs, at the same time feces are suitable materials for detection of virus in the enteric form exactly for the same reasons. Recently, a new technique of in vitro DNA amplification, Known as the polymerase chain reaction (PCR), has been widely applied to clinical viral diagnosis because of its sensitivity, specificity and rapidity. In this research, we attemped to set up the PCR for the detection of CPV in fecal samples and conformed the canine parvpviral enteritis by PCR. To increase the sensitivity and specificity of a PCR, the nested PCR (two-step PCR) was performed. We also surveyed the contamination status of CPV in the research using fecal specimen was highly sensitive and specific. Of the 100 fecal specimens suspected canine parvoviral enteritis, 45 fecal specimens were positive in HA test, 64 fecal specimens were positive in the first PCR, and 87 fecal specimens were positive in the second PCR. CPV contamination status of animal clinics and breeding centers was serious, wo hygienic management of environment in which dogs are reared is required. The nested PCR described here seems to be a rapid, sensitive and specific for the detection of canine parvovirus.

• Journal of Veterinary Science
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• v.21 no.5
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• pp.71.1-71.10
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• 2020

Background: Feline calicivirus (FCV) is a major and highly infectious pathogen in cats worldwide. However, there have been limited studies about the status of FCV infections in Korea. Objectives: To investigate the current status of FCV infections in stray cats in Korea. Methods: A novel reverse transcription polymerase chain reaction (RT-PCR) assay was developed based on the conserved nucleotide sequences of reported FCV strains. Field swab samples were collected from 122 cats (2 hospital admitted cats and 120 stray cats) in 2016 and 2017. All the samples were tested by virus isolation and 2 different RT-PCRs, including the novel RT-PCR, for the detection of FCV. Results: The novel RT-PCR assay showed no cross-reactivity to the nucleic acids of the other feline pathogens tested, and the limit of detection was calculated as 10⁰ TCID₅₀/mL based on an in vitro assessment. The novel RT-PCR assay detected 5 positive samples from the 122 field samples, which showed perfect agreement with the results of the virus isolation method. In contrast, another RT-PCR assay used in a previous study in Korea detected no positive samples. The prevalence of FCV infection in stray cats was 2.5% (3/120) based on the results of virus isolation and the novel RT-PCR assays. Conclusions: The current study is the first report of the detection and prevalence of FCV in stray cats in Korea. The novel RT-PCR assay developed in this study showed high sensitivity and specificity, which indicates a useful diagnostic assay to identify FCV infection in cats.

• The Korea Journal of Herbology
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• v.29 no.3
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• pp.19-26
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• 2014

Objectives : Aucklandiae Radix (Muxiang) one of important herbal medicines in oriental medicine, is defined as the dried root of Aucklandia lappa (Asteraceae). Owing to the similarities in the morphology and name, Inulae Radix (Tu-Muxiang) and Vladimiriae Radix (Chuan-Muxiang) as well as Aristolochiae Radix (Qing-Muxiang) originated from other medicinal plants are often used as substitutes and/or adulterants of Aucklandiae Radix. Therefore, a reliable authentication of these herbal medicines is necessarily for the public health and prevention of misuse. Methods : 32 samples of medicinal plants supplying Aucklandiae Radix, Inulae Radix, Vladimiriae Radix, and Aristolochiae Radix were collected in Korea and China. The ITS (Internal transcribed spacer) nucleotide sequences of samples were determined. The PCR primers to amply DNA marker of each herbal medicine were designed basing on the specific ITS regions showing differences in the sequences among medicinal plants. Results : Primer set Al R/IS F designed in this work amplified 220 bp PCR product only in samples of Aucklandiae Radix. In contrast, primer set Ih F/IS R, Vs R/IS F, and AcR F1/Ac R amplified 250 bp product, 356 bp prouct, and 516 bp product respectively to identify Inulae Radix, Vladimiriae Radix, and Aristolochiae Radix. Conclusions : The primers designed basing on the nucleotide sequences of ITS regions appearing differenced in the sequences among medicinal plants amplified the DNA markers for the identification of Aucklandiae Radix, Inulae Radix, Vladimiriae Radix, and Aristolochiae Radix. These herbal medicines were more efficiently identified by multiplex PCR method using all primers in a single PCR process.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2005.09a
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• pp.147-150
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• 2005

Proper PCR primer design determines the success or failure of Polymerase Chain Reaction (PCR) reactions. In this project, we develop GENE-PRIMER, a genomes specific PCR primer design program that is amenable to a genome-wide scale. To achieve this, we incorporated various parameters with biological significance into our program, namely, primer length, melting temperature of primers Tm, guanine/cytosine (GC) content of primer, homopolymeric runs in primer and self-hybridization tendency of primer. In addition, BLAST algorithm is utilized for the purpose of primer specificity check. In summary, selected primers adhered to both physico-chemical criteria and also display specificity to intended binding site in the genome.

• The Plant Pathology Journal
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• v.17 no.6
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• pp.329-335
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• 2001

Yellowing disease of palms caused by phytoplasma is spreading in the Arabian Gulf region. Surveys were conducted to determine the occurrence of the disease. Electron and fluorescence microscopy, and polymerase chain reaction (PCR) techniques were used to detect the phytoplasma associated with the yellowing disease of ornamental palm Washingtonia sp. grown in Kuwait. An accumulation of phytoplasmal DNA was observed by fluorescence microscopy in phloem tissues of diseased palms. Electron microscopy showed that phytoplasma cells were primarily confined to the phloemsieve elements of tissue samples collected from infected mature palms in the field. The pathogen was identified on the basis of molecular analysis using universal and specific nested primers in PCR amplifications. Prokaryotic 16S rDNA gene was detected in amplified PCR products. Nested PCR resulted in DNA amplification of 1.2 kbp fragment. This is the first report of a phytoplasmal rDNA gene identified from the putative causal pathogen of yellows in ornamental palms in the Arabian Gulf region.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.3
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• pp.273-276
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• 2006

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of lower organisms. Recently, we have reported that a transfection of human U6 promoter (hU6) driven hairpin small-interference RNA (siRNA) plasmid specifically knocks down the target gene by post-transcriptional gene silencing in mammalian cells. Here we report that transfection of polymerase chain reaction (PCR) products, containing human U6 promoter with hairpin siRNA, knocks down the target gene expression in human teratocarcinoma NT-2/D1 cells. Moreover, we showed 3' end termination sequence, 5 Ts, is not critical elements for knocking down in PCR-based siRNA system. Therefore, the PCR-based siRNA system is a promising tool not only for the screening but also to temporally regulate gene expression in the human progenitor cells.

• Korean Journal of Veterinary Service
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• v.23 no.3
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• pp.227-233
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• 2000

Salmonella enteritidis is the most prevalent etiologic agents of foodborne acute gastroenteritis. Direct isolation and identification of S enteritidis are time consuming work and not so highly sensitive. This study was conducted to develop for the specific detection of S enteritidis using polymerase chain reaction(PCR). PCR primers were selected to amplify a 351-base pair(bp) DNA fragment from the salmonella plasmid virulence A(spv A) gene of S enteritidis. With the primers, 351 bp DNA products were amplified from S enteritidis but not from other B, D, Cl serogroup Salmonella spp. It was sensitive to detect up to 40 pg of template DNA by agarose gel electrophoresis. This PCR assay is very rapid and specific method and less time consuming than the standard bacteriological methods.

• Korean Journal of Veterinary Service
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• v.39 no.4
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• pp.247-251
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• 2016

Salmonella is one of the most common bacteria that causes heavy losses in swine industry and major causative pathogen of food poisoning in public health. Various methods for the identification of Salmonella such as Gram staining, agglutination test, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) have been used. Several studies have demonstrated that Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI TOF) Mass Spectrometry (MS) identification is an efficient and inexpensive method for the rapid and routine identification of isolated bacteria. In this study, MALDI TOF MS could provide rapid, accurate identification of Salmonella spp. from swine compared with end point PCR and real time PCR.