• Korean Journal of Veterinary Research
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• v.37 no.2
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• pp.349-358
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• 1997

Mycobacterium paratuberculosis is the etiologic agent of Johne's disease, a chronic inflammatory bowel syndrome in ruminants. The attempts to control or eradicate the disease were severely hampered by the inadequacies of present diagnostic methods. The first purpose of this study was to detect Johne's disease out of 577 cows in the province of Kyunggi, Chungchong, Gangweon and the second purpose was to compare the results of non-absorbed ELISA, absorbed ELISA, PCR, and conventional culture methods. The third purpose was to increase diagnostic specificity, accuracy and rapidity. When non-absorbed ELISA test was conducted with Mycobacterium paratuberculosis antigen, the prevalence of positive was 10.9%. To increase diagnostic specificity, absorbed ELISA test with Mycobacterium phlei was used. In this test, the positive prevalence was 1.7%. For the specific detection of Mycobacterium paratuberculosis, PCR was applied to bacterial culture obtained from fecal samples of cattle. The DNA sequences derived from IS900 were used to prepare DNA primers for detection and identification of Mycobacterium paratuberculosis by PCR. PCR for M paratuberculosis isolated from fecal cultures amplified specific target DNA. PCR was much more rapid than that obtained by conventional culture technique in diagnosis of Johne's disease.

• Journal of Life Science
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• v.20 no.6
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• pp.955-959
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• 2010

Differential diagnosis of the taeniid proglottids between Taenia asiatica and T. sagniata is a daunting task due to their close morphological similarity. However, to correctly diagnose them on time is important in managing infected patients, as well as for reducing serious complications such as cysticercosis. Currently, DNA-based methods for the dissection of genomic information of parasites are being employed to make accurate and rapid diagnoses in the field of parasitology. In this study, multiplex PCR was established and exploited to identify exact species of taeniid adult worms recovered from Korean people. To discriminate one from the other other, primers-Ta4978F, Ts5058F, Tso7421F, and Rev7915- were used for the multiplex PCR, which provided swift and precise identification of the taeniid worms being observed. Also,having instituted PCR methodology, we ascertained that easiness would be achieved to reassess and re-evaluate Korean endemic data on human taeniid cestodes.

• Journal of Veterinary Clinics
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• v.17 no.1
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• pp.13-20
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• 2000

Recently cases of canine distemper have occurred in Korea despite vaccination was carried out nationwidely. This study was performed to establish rapid diagnosis of canine distemper by RT-PCR, nested PCR, and serological test. A total of 30 dogs, which were suspected canine distemper clinically, was examined. RT-PCR and nested PCR were specific for the amplification of CDV H gene and sensitive to detect 7 TCID50 of Onderstepoort strain. By RT-PCR, H gene was detected in 6(20%) of 30 peripheral bloods from dogs. And H gene was detected in 10(33.3%) of 30 samples by nested PCR. H gene was detected from 1 brain of 6 years-old Beagle dog and 1 lung of 2 months-old Shihtzu dog, in which peripheral blood H gene was not detected. Serum neutralizing antibody titer against Onderstepoort strain ranged from 4 to 1,024 in 30 patients. No correlation was observed between the results of nested PCR and titiers of neutralizing antibody.

• Tuberculosis and Respiratory Diseases
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• v.76 no.4
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• pp.163-168
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• 2014

Background: Mycobacterial identification in active pulmonary tuberculosis (APTB) is confirmative, even though successful rates using self-expectorated sputum are limited. Sputum specimens collected by hypertonic saline nebulization showed higher bacteriologic diagnostic sensitivities over those of self-expectoration, mostly studied in smear-negative or sputum-scarce patients. The efficacy of induced sputum was rarely assessed in real clinical settings. Methods: A prospective randomized case-control study was performed in one hospital. The subjects highly suspicious of APTB were asked to provide 3 pairs of sputum specimens in 3 consecutive days. The first pairs of the specimens were obtained either by self-expectoration (ES) from the next day of the visit or sputum induction with 7% saline nebulization in clinic (SI), and the other specimens were collected in the same way. The samples were tested in microscopy, culture, and polymerase chain reaction (PCR). The outcomes of the bacteriological diagnosis were compared. Results: Seventy six patients were assigned to either ES (38 subjects, median age of 51, 65.8% male) or SI (38 subjects, median age of 55, 52.6% male). APTB was clinically confirmed in 51 patients (70.8%), 27 in ES and 24 in SI. Among the APTB, more adequate specimens were collected from SI (41/65, 63.1%) than ES (34/80, 42.5%) (p=0.01). Bacteriological confirmation was achieved in 14 (58.3%) patients in SI, and 13 (48.1%) in ES (p=0.46). In the same-day bacteriological diagnosis with microscopy and PCR, there were positive results for 9 patients (37.5%) in SI and 7 patients (25.9%) in ES (p=0.37). Conclusion: Sputum induction improves sputum specimen adequacy. It may be useful for the same-day bacteriological diagnosis with microscopic examination and PCR.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.345-352
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• 1998

The diagnosis of brucellosis is currently based on serological and microbiological tests. However, the microbiological isolation and identification have several disadvantages such as time-consuming and laborious, and the serological methods have been reported to cross-react with antigens other than those from Brucella spp. To develop a sensitive and rapid diagnostic method for detection of Brucella species, the genus-specific primers were designed and synthesized from the sequence of gene encoding a 31kDa cell surface protein(BCSP) and a 36kDa outer membrane protein(OMPB) of B abortus. The amplified 711bp and 982bp DNA fragments were only visible in each species of Brucella by PCR method using the BCSP and OMPB primers, respectively. However, PCR product was not obtained with DNA from other Gram-negative bacteria. As little as 1pg of the B abortus genomic DNA could be detected by this PCR method. Using the PCR technique, semen samples from 185 bulls of Brucella-seronegative herds in Cheju island were examined for comparison of this PCR method with conventional methods in 1995. The semen samples from 5 bulls were positive by culture method and PCR, and one was positive and 5 were suspect by semen plasma agglutination test. However, the semen samples obtained from 177 bulls were negative by semen plasma agglutination, culture and PCR methods in 1996. The results of comparison tests suggested that PCR was a better test than agglutination test against semen of bulls. This study indicated that the PCR technique was a valuable for the diagnosis of bovine brucellosis, particulary in bull semens.

• Biomedical Science Letters
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• v.25 no.4
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• pp.426-430
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• 2019

Currently, molecular diagnostic assays based on nucleic acid amplification tests have been shown to effectively detect mycobacterial infections in various types of specimen, however, variable sensitivity was shown in FFPE samples according to the kind of commercial kit used. The present study therefore used automated PCR-reverse blot hybridization assay (REBA) system, REBA Myco-ID HybREAD 480^®, for the rapid identification of Mycobacterium species in various types of human tissue and compared the conventional one-tube nested-PCR assay for detecting Mycobacterium tuberculosis (MTB). In conventional nested-PCR tests, 25 samples (48%) were MTB positive and 27 samples (52%) were negative. In contrast, when conducted PCR-REBA assay, 11 samples (21%) were MTB positive, 20 samples (39%) were NTM positive, 8 samples (15%) were MTB-NTM double positive, and 13 samples (25%) were negative. To determine the accuracy and reliability of the two molecular diagnostic tests, the one-tube nested-PCR and PCR-REBA assays, were compared with histopathological diagnosis in discordant samples. When conducted nested-PCR assay, 10 samples (59%) were MTB positive and seven samples (41%) were negative. In contrast, when conducted PCR-REBA test, three samples (17%) were MTB positive, 10 samples (59%) were NTM positive and four samples (24%) were negative. In conclusion, the automated PCR-REBA system proved useful to identify Mycobacterium species more rapidly and with higher sensitivity and specificity than the conventional molecular assay, one-tube nested-PCR; it might therefore be the most suitable tool for identifying Mycobacterium species in various types of human tissue for precise and accurate diagnosis of mycobacterial infection.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.112-117
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• 2013

Wheat streak mosaic virus (WSMV), a member of the genus Tritimovirus in Potyviridae, severely impacts wheat and corn seed worldwide, but has yet to be detected in Korea, and hence, every effort should be made to prevent its introduction. To prevent WSMV from entering the country, it is necessary to prepare a specific, sensitive, simple, and fast detection method for routine application to plant quarantine procedures. For this reason, a two-step diagnosis system consisting of RT-PCR and nested PCR is being used for WSMV detection. In addition, a novel positive control was developed for use with the system. WSMV has been detected in seed sweet corn from Japan and seed wheat from USA by a two-step diagnosis system, the details of which are described in this study. After sequence analysis, similarities of 80.6 and 100.0% with other isolates were determined by BLAST. They showed the same topology, which was classified as 4 genotypes by various phylogenetic trees, using a poly protein encoding sequence amplification. In this analysis, WSMV-JSweet-corn2868 (JX845574) is classified as clade B, while WSMV-Uwheat1944-1 (KC754959) and WSMV-Uwheat1944-2 (KC754960) belong to clade D.

• Research in Plant Disease
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• v.7 no.3
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• pp.123-133
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• 2001

Many pathovars of the species Pseudomonas syringae are known to produce different phytotoxins as secondary metabolites. Although phytotoxins generally enhance the virulence of P. syringae, they are not required for pathogenesis. Among the phytotoxins produced by P. syringae, lipodepsipeptides, coronatine, phaseolotoxin, and tabtoxin are the most well-known toxins which have been intensively studied for their structure, mode of action, biosynthesis, and regulation. A polymerase chain reaction (PCR) technique that amplifies a segment of the phytotoxin gene cluster using a primer set has been developed in recent years. This method offers the advantages of speed and sensitivity compared to the approaches based on physiological and biochemical methods. PCR detection of genes involved in the production of toxins could be exploited for early diagnosis of plant diseases caused by P. syringae pathovars.

• Journal of Genetic Medicine
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• v.17 no.1
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• pp.27-33
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• 2020

Purpose: Duchenne muscular dystrophy (DMD) is the most common lethal muscular dystrophy and is caused by the genetic variants of DMD gene. Because DMD is X-linked recessive and shows familial aggregates, prenatal diagnosis is an important role in the management of DMD family. We present our experience of prenatal molecular diagnosis and carrier detection based on multiplex polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA), and linkage analysis. Materials and Methods: During study period, 34 cases of prenatal diagnosis and 21 cases of carrier detection were performed at the Seoul National University Hospital. Multiplex PCR and MLPA was used to detect the exon deletions or duplications. When the DMD pathogenic variant in the affected males is unknown and no DMD pathogenic variant is detected in atrisk females, linkage analysis was used. Results: The prenatal molecular diagnosis was offered to 34 fetuses. Twenty-five fetuses were male and 6 fetuses (24.0%) were affected. Remaining cases had no pathogenic mutation. We had 24 (80.0%) cases of known proband results; exon deletion mutation in 19 (79.2%) cases and duplication in 5 (20.8%) cases. Linkage analysis was performed in 4 cases in which 2 cases (50.0%) were found to be affected. In the carrier testing, among 21 cases including 15 cases of mother and 6 cases of female relative, 9 (42.9%) cases showed positive results and 12 (57.1%) cases showed negative results. Conclusion: Prenatal molecular diagnosis and carrier detection of DMD are effective and feasible. They are useful in genetic counseling for DMD families.

• Korean Journal Plant Pathology
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• v.12 no.3
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• pp.358-362
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• 1996

감자 잎말림 바이러스를 검정하기 위하여 ELISA 및 전자현미경에 의해 바이러스 감염이 확인된 기내 배양중인 감장의 줄기로부터 RT-PCR 분석을 수행하였다. 분리된 총 RNA들로부터 바이러스 cDNA를 합성하고 감자 잎말림 바이러스 외피단백질의 일부인 465bp를 특이하게 증폭하도록 고안한 두 primer를 사용하여 PCR 반응을 하였다. 증폭된 465pb의 DNA 절편의 염기서열을 분석한 결과 역시 감자 잎말림 바이러스임을 확인하였다. 바이러스 검정에 있어서 EL-ISA 방법과 RT-PCR 방법간의 민감도를 조사한 결과 RT-PCR 방법간의 민감도를 조사한 결과 RT-PCR 방법이 ELISA 방법보다 감자 잎말림 바이러스검정에 있어서보다 정확한 방법인 것으로 사료된다.