• 대한의생명과학회지
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• 제16권2호
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• pp.127-131
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• 2010

Mycobacterium leprae detection is difficult even with molecular biological techniques due to the low sensitivity of current methodologies. In this report, real-time PCR targeting the M. leprae-specific repetitive element (RLEP) sequence was developed as a new diagnostic tool and evaluated using clinical specimens. For this, M. leprae DNAs were extracted from skin biopsy specimens from 80 patients and analyzed by real-time PCR using TaqMan probe. Then, the detection efficiency of the real-time PCR was compared with that of standard PCR. In brief, the rate of positive detection by the standard PCR and real-time PCR was 32.50% and 66.25%, respectively. The results seemed to clearly show that the TaqMan real-time PCR developed in this study may be a useful tool for sensitive detection of M. leprae from clinical specimens.

• The Plant Pathology Journal
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• 제30권4호
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• pp.440-444
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• 2014

Iris yellow spot virus (IYSV) is a plant pathogenic virus which has been reported to continuously occur in onion bulbs, allium field crops, seed crops, lisianthus, and irises. In South Korea, IYSV is a "controlled" virus that has not been reported, and inspection is performed when crops of the genus Iris are imported into South Korea. In this study, reverse-transcription polymerase chain reaction (RT-PCR) and nested PCR inspection methods, which can detect IYSV, from imported crops of the genus Iris at quarantine sites, were developed. In addition, a modified positive plasmid, which can be used as a positive control during inspection, was developed. This modified plasmid can facilitate a more accurate inspection by enabling the examination of a laboratory contamination in an inspection system. The inspection methods that were developed in this study are expected to contribute, through the prompt and accurate inspection of IYSV at quarantine sites to the plant quarantine in South Korea.

• Plant Resources
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• 제2권2호
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• pp.69-74
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• 1999

A rapid and sensitive assay for specific detection and identification of barley yellow mosaic virus(BaYMV) was set up using the reverse transcriptase polymerase chain reaction(RT-PCR). A couple of primers was select to discriminate the viruses. PCR fragments of BaYMV(ca.0.9 kb) were obtained by using the method designed for BaYMV capsid protein. RT-PCR fragments were cloned with vector pT7 Blue and the resulting clones were sequenced. Capsid protein of BaYMV consisted of 297 amino acids and 891 nucleotides. The capsid protein sequence of BaYMV showed that 98% of nucleotides and 99% of amino acids homology.

• 한국동물위생학회지
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• 제37권4호
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• pp.253-261
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• 2014

Porcine circovirus disease (PCVD) is a major problem of swine industry worldwide, and diagnosis of PCV2, causal agent of PCVD, has been doing in clinical laboratories of pig disease by polymerase chain reaction (PCR) methods. But the PCR analyses have a serious problem of misdiagnosis by contamination of DNA, in particular, from carryover contamination with previously amplified DNA or extracted DNA from field samples. In this study, an uracil DNA glycosylase (UNG)-based direct PCR (udPCR) without DNA extraction process and DNA carryover contamination was developed and evaluated on PCV2 culture and field pig samples. The sensitivity of the udPCR combined with dPCR and uPCR was same or better than that of the commercial PCR (cPCR) kit (Median diagnostics, Korea) on PCV2-positive serum, lymph node and lung samples of the pigs. In addition, the udPCR method confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PCV2 DNA from previous udPCR. In clinical application, 170 pig samples (86 tissues and 84 serum) were analysed by cPCR kit and resulted in 37% (63/170) of positive reaction, while the udPCR was able to detect the PCV2 DNA in 45.3% (77/170) with higher sensitivity than cPCR. In conclusion, the udPCR developed in the study is a time, labor and cost saving method for the detection of PCV2 and providing a preventing effect for DNA carryover contamination that can occurred in PCR process. Therefore, the udPCR assay could be an useful alternative method for the diagnosis of PCV2 in the swine disease diagnostic laboratories.

• Fisheries and Aquatic Sciences
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• 제8권3호
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• pp.122-127
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• 2005

A DNA chip that rapidly and accurately detects the viral genes in rhabdovirus-infected fish was developed. The N, Ml, and G proteins of three rhabdovirus strains, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), and flounder rhabdovirus (HIRRV), were selected for use as probes. The sequences of the corresponding genes were obtained, and probes were prepared by PCR using specific primer sets. The specificity of the probes was confirmed by cross PCR. The prepared probes were spotted on poly-L-lysine- or aminosilane-coated glass slides and hybridized with target DNA under several different conditions in order to determine the optimal hybridization temperature, glass-slide coating, and target cDNA concentration.

• Parasites, Hosts and Diseases
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• 제54권3호
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• pp.323-327
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• 2016

A man with only yellowing of the skin and eye sclera was diagnosed with clonorchiasis, which rarely manifested jaundice as the initial symptom. However, because of a lack of evidence for a diagnostic gold standard, the time until definitive diagnosis was more than a week. The diagnostic process relied on inquiring about the patient's history, including the place of residence, dietary habits, and symptoms, as well as on serological findings, an imaging examination, and pathological findings. MRCP and CT results showed mild dilatation of intrahepatic ducts and increased periductal echogenicity. The eggs were ultimately found in stool by water sedimentation method after the negative report through direct smear. DNA sequencing of PCR production of the eggs demonstrated 98-100% homology with ITS2 of Clonorchis sinensis. After anti-parasite medical treatment, the patient's symptoms were gradually relieved. Throughout the diagnostic procedure, besides routine examinations, the sedimentation method or concentration method could be used as a sensitive way for both light and heavy C. sinensis infection in the definite diagnosis.

• Pediatric Infection and Vaccine
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• 제3권2호
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• pp.207-213
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• 1996

Herpes simplex virus(HSV) infections of the CNS are associated with significant morbidity and mortality even when appropriate antiviral therapy is administered. HSV infections of the brain can be subdivided into two categories : neonatal HSV infections, which usually are caused by HSV type 2, and herpes simplex encephalitis(HSE), which occur in patients over 3 months old and is nearly uniformly caused by HSV type 1. The clinical presentation of HSE is one of the focal encephalopathic process associated with altered levels of consciousness, fever, focal seizures and hemiparesis. But because of the lack of pathognomic clinical presentation and diagnostic procedure, the efforts to develop alternative diagnostic procedure have led to the use of new diagnostic technique such as polymerase chain reaction(PCR). We report a case of HSV type 1 encephalitis in 13 month old male infant who presented with altered level of consciousness, fever and focal seizures. With the use of the PCR, HSV-1 DNA was detected in cerebrospinal fluid from the patient. The symptoms and signs of encephalitis subsided by treatment with acyclovir in 14 days.

• 대한수의학회지
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• 제58권1호
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• pp.27-31
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• 2018

Canine coronavirus is a single-stranded RNA virus that causes enteritis in dogs of any age. Coronaviral enteritis is seldom definitively diagnosed, since it is usually much less severe than many other types of enteritis and is self-limiting. Conventional diagnostics for the canine coronaviral enteritis such as polymerase chain reaction (PCR), virus isolation, and electron microscopic examination are inappropriate for small animal clinics due to the complicated experimental processes involved. Therefore, a commercially available lateral flow test kit based on chromatographic immunoassay techniques was tested to evaluate its performance as a first-line diagnostic test kit that could be used in clinics. The coronavirus antigen test kit detected canine coronavirus-infected dogs with 93.1% sensitivity and 97.5% specificity. The detection limit of the test kit was between $1.97{\times}10^4/mL$ and $9.85{\times}10^3/mL$ for samples with a 2-fold serial dilution from $1.25{\times}10^6\;TCID_{50}$ ($TCID_{50}$, 50% tissue culture infectious dose). Additionally, the test kit had no cross-reactivity with canine parvovirus, distemper virus, or Escherichia coli. Overall, the commercially available test kit showed good diagnostic performance in a clinical setting, with results similar to those from PCR, confirming their potential for convenient and accurate use in small animal clinics.

• 농업과학연구
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• 제47권1호
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• pp.173-182
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• 2020

Human Astrovirus (HuAstV), known as a waterborne virus, is a group IV positive-sense single-stranded RNA that belongs to Astroviridae. The first outbreak of HuAstV was reported in England in 1975. HuAstV can exist not only among clinical patients but also in various water environments, such as water for agriculture and vegetables. For diagnosis of HuAstV from water samples, a polymerase chain reaction (PCR) system has been developed. However, the PCR-based diagnostic method has problems in field application, such as reaction time, sensitivity and specificity. For this reason, in this study we developed the loop-mediated isothermal amplification assay (LAMP) system, aimed specifically at HuAstV. Three prepared LAMP primer sets were tested by specificity, non-specificity and sensitivity; one LAMP primer set was selected with optimum reaction temperature. The developed LAMP primer set reaction conditions were confirmed at 62℃, and detection sensitivity was 1 fg/μL. In addition, restriction enzyme HaeIII (GG/CC) was introduced to confirm that the LAMP reaction was positive. As a result, selected LAMP primer set was 100 - 1000 times more specific, rapid, and sensitive than conventional-nested PCR methods. For verification of the developed LAMP assay, twenty samples of cDNA from groundwater samples were tested. We expect that the developed LAMP assay will be used to diagnose HuAstV from various samples.

• 대한수의학회지
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• 제42권3호
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• pp.351-362
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• 2002

Newcastle disease (ND) is a highly contagious infection of poultry, Two pathology-based techniques, in situ RT-PCR and in situ hybridization (ISH) were applied to formalin-fixed, paraffin-embedded tissues from chickens naturally infected with velogenic ND virus (VNDV). Two pairs of primers and a probe for ISH and in situ RT-PCR, respectively, were selected from highly conserved region of matrix gene of NDV. The ISH experiment was carried out using MicroProbe$^{TM}$ capillary action system within 2 hours. In situ RT-PCR was performed using MicroProbe$^{TM}$ capillary action system and GeneAmp In Situ PCR system. With ISH and in situ RT-PCR, viral nucleic acid was detected in the central nervous system of chickens from infected with neurotropic velogenic Newcastle disease virus (NVNDV), whereas viral nucleic acid was detected in various organs or tissues of chickens from infected with viscerotropic velogenic Newcastle disease virus (VVNDV). In the NVND group, positive signals were characteristically defined in the cytoplasm of neuron, vascular endothelial cells, and perivascular mononuclear macrophages in the central nervous system. One of NVND group, chicken from one farm exhibited positive signals in the bronchial epithelium. The VVND group chickens showed positive reaction in the macrophages, vascular endothelium, and bronchiolar epithelium. Markedly, viral nucleic acid was detected in the macrophages of morphologically normal tissues which were peripheral or located in distant areas from lesions. The central nervous system of chickens infected with VVND virus had positive signals in the vascular endothelial cell, perivascular mononuclear macrophages and some neuron. The number and intensity of the positive cells by in situ RT-PCR were more and stronger, respectively, in comparison with those by ISH. Particularly, positive reaction was detected in macrophages infiltrating in cardiac muscle by in situ RT-PCR, but not obtained by ISH. Therefore, these results demonstrated that ISH is a rapid diagnostic method for detection of NDV and in situ RT-PCR can be used as an efficient method for detection of low viral load infection or subclinical viral infection of NDV.